Heterologous gene silencing induced by tobacco rattle virus (TRV) is

ORIGINAL PAPER

Heterologous gene silencing induced by tobacco rattle virus (TRV)is ef?cient for pursuing functional genomics studies in woody plants

Yuanzhong Jiang ?Shenglong Ye ?Lijun Wang ?

Yanjiao Duan ?Wanxiang Lu ?Hong Liu ?Di Fan ?Faqi Zhang ?Keming Luo

Received:26June 2013/Accepted:11October 2013óSpringer Science+Business Media Dordrecht 2013

Abstract Virus-induced gene silencing (VIGS)is an effective tool for studying the functions of plant genes,but only a few VIGS vectors available for woody plants were reported so far.Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV)vectors.We ?rst tested whether the TRV-vector can be directly applied to infect woody plant species,such as Vernicia fordii ,Populus tomentosa Carr.and Camellia oleifera.The results revealed that TRV-mediated VIGS could be effectively elicited in V.fordii ,weakly in P.tomentosa Carr.,but not in C.oleifera.TRV-based VIGS vectors with heterologous phytoene desaturase (PDS )sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benth-amiana and V.fordii .The photobleached leaf phenotype of silenced plants signi?cantly correlated with the down-reg-ulation of endogenous PDS as compared with controls.To further con?rm the reliability of VIGS in V.fordii ,we also isolated the cloroplastos alterados 1gene from P.tomen-tosa Carr.,and the silencing pheotypes of albino leaves were observed in V.fordii 2weeks after inoculation using a heterologous TRV-based VIGS system.Taken together,we have successfully developed an Agrobacterium -mediated VIGS assay in V.fordii and demonstrated that V.fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.Keywords Virus-induced gene silencing á

Functional genomics áVernicia fordii áPopulus áNicotiana benthamiana áCamellia oleifera

Introduction

Since the whole-genome sequence of Populus trichocarpa was released in 2006(Tuskan et al.2006),whole genome sequencing has been performed widely in woody plants,including grapevine (Scalabrin et al.2010),papaya (Yu et al.2009),pear (Wu et al.2013)and apple (Pagliarani et al.2012).With the availability of the whole-genome sequences of increasing woody plant species,it is essential to develop novel molecular tools for large-scale analysis of gene functions at the genome-wide level.Ultimately,understanding of the molecular mechanisms of gene function and regulation will enable biomass improvement in woody plants by genetic engineering and molecular breeding.

Yuanzhong Jiang and Shenglong Ye have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11240-013-0393-0)contains supplementary material,which is available to authorized users.

Y.Jiang áS.Ye áL.Wang áY.Duan áH.Liu áD.Fan áF.Zhang áK.Luo (&)

Key Laboratory of Eco-environments of Three Gorges Reservoir Region,Ministry of Education,Chongqing Key Laboratory of Transgenic Plant and Safety Control,Institute of Resources Botany,School of Life Sciences,Southwest University,No.1,Tiansheng Road,Beibei,Chongqing 400715,China e-mail:luokeming@https://www.sodocs.net/doc/185449689.html,

W.Lu

College of Horticulture and Landscape Architecture,Southwest University,Chongqing 400716,China

W.Lu áF.Zhang áK.Luo

Key Laboratory of Adaptation and Evolution of Plateau Biota,Northwest Institute of Plateau Biology,Chinese Academy of Sciences,Xining 810008,China

Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-013-0393-0

There are various large-scale approaches,including chemical mutagenesis,irradiation mutagenesis,post-tran-scriptional gene silencing(PTGS),T-DNA-and transpo-son-based insertional mutant populations,to introduce mutations or silence of gene expression(Martienssen1998; Waterhouse et al.1998;Ostergaard and Yanofsky2004; Robinson and Parkin2009).Although these approaches have been applied successfully to study of gene functions at the genome-wide level for other model plants such as Arabidopsis thaliana,large collections of mutant popula-tions is impractical to apply in woody plants due to the technical challenge of transformation,long growth cycle and large genome size.In addition,these functional genomic approaches when used in trees have also other substantial obstacles,such as lack of obvious phenotypes due to the presence of large gene families,the large pop-ulations required to disrupt a gene of interest,gene tar-geting complications from multiple mutations or insertions and gene duplication by polyploidy and lethality with complete loss of gene function(Borevitz and Ecker2004; Candela et al.2011).

Virus-induced gene silencing(VIGS)is an extremely powerful tool for large-scale functional analysis of indi-vidual gene by silencing the expression of endogenous genes(Baulcombe1999;Dinesh-Kumar et al.2003;Lu et al.2003;Burch-Smith et al.2004).This technique was developed based on the RNA-mediated PTGS that func-tioned as a natural defense system against various viruses in plants and other organisms(Hamilton and Baulcombe 1999;Dinesh-Kumar et al.2003;Burch-Smith et al.2004; Robertson2004).Double stranded RNA(dsRNA)is of importance in the VIGS process.The dsRNAs is sheared into siRNAs whose length is from21to23nt.The siRNAs can integrate into be RNA-induced silencing complexes (RISC)and then target their homologous mRNA for cleavage,which spells the degradation of the targeted mRNA(Robertson2004).By this mean,it is theoretically possible to knockdown almost any gene of interest if a suitable virus vector is used for the plant species under investigation.This approach also avoids the need for plant transformation and overcomes the problems of functional redundancy(Burch-Smith et al.2004;Becker and Lange 2010).

With advantages of convenience,rapidity and ef?-ciency,the VIGS technology achieves success for a wide range of herbaceous plants tobacco(Nicotiana benthami-ana),tomato(Solanum lycopersicum),potato(Solanum tuberosum),Petunia hybrida,pea(Pisum sativum),Arabi-dopsis thaliana,rice(Oryza sativa),maize(Zea mays), barley(Hordeum vulgare),soybean(Glycine max),cassava (Manihot esculenta),pepper(Capsicum annuum),and wheat(Triticum aestivum)(Benedito et al.2004;Burch-Smith et al.2004;Chung et al.2004;Constantin et al.2004;Faivre-Rampant et al.2004;Fofana et al.2004; Robertson2004;Ding et al.2006;Wang et al.2006;Zhang and Ghabrial2006;Doja and Koonin2013).Unfortunately, VIGS is not effective in many other plant species,espe-cially in trees,due to the lack of a compatible VIGS vector (Robertson2004).To date,there are only a few studies on effective VIGS-inducing virus vectors that are able to be used for woody plant species.These viruses include poplar mosaic virus(PopMV)(Naylor et al.2005),Plum pox virus (Lansac et al.2005),apple latent spherical virus(ALSV) (Sasaki et al.2011)and Grapevine leafroll-associated virus-2(GLRaV-2)(Kurth et al.2012).However,it is still unknown whether these vectors are effective VIGS inducers and can be used for analysis of gene functions in other woody plants.Therefore,a high-throughput VIGS-mediated method for assessing gene function is not yet available for most of woody plant species.

Previously,it has been demonstrated that heterologous VIGS is a promising approach for functional genomic analysis in model plants as long as there is minimal nucleotide sequence homology between the gene sequen-ces(Ekengren et al.2003;Fofana et al.2004;Ryu et al. 2004;Senthil-Kumar and Udayakumar2006).For instance, a fragment of phytoene desaturase(PDS)gene,encoding an enzyme required for the biosynthesis of carotenoid pigments that protect chlorophyll from photobleaching, from a monocot(Lilium longi?orum)caused the silencing of endogenous PDS in N.benthamiana by VIGS in spite of the remote evolutionary relationship between these two species(Benedito et al.2004).The endogenous PDS genes of tomato,N.tabacum and Petunia hybrida were suc-cessfully silenced using the N.benthamiana PDS gene sequence(Ryu et al.2004).A late embryogenic abundant-4 (lea4)gene from peanut(Arachis hypogaea)was used to ef?ciently silence its ortholog in tomato(Senthil-Kumar and Udayakumar2006).Similarly,a DEAD box helicase gene from Dunaliella salina was used to silence its ortholog in a distantly related species,N.benthamiana (Howes and Kumagai2005).A recent study has demon-strated that tobacco rattle virus(TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-ef?cient plant N.benthamiana (Senthil-Kumar et al.2007).However,it is still unknown whether TRV-mediated VIGS is an effective genetic tool for rapid assessment of plant gene functions in woody species,especially when heterologous gene sequences are used.

TRV-mediated VIGS has been commonly used in many plants,including dicots and monocots(Burch-Smith et al. 2004;Brigneti et al.2004;Becker and Lange2010).The system has also been successfully applied in some woody

Plant Cell Tiss Organ Cult

plant species.For example,Ye et al.(2009)reported that TRV-based vector could trigger virus-induced gene silencing in Jatropha curcas L.,which is a small tropical, woody tree belonging to the Euphorbiaceae family. Recently,an Agrobacterium-mediated VIGS assay was successfully developed in several cotton cultivars with various genetic backgrounds(Gao et al.2011).Therefore, TRV is an alternative tool for functional genomic analyses of many tree species by VIGS.To extend the use of TRV-VIGS vector to diverse woody plant species,in this study, we?rst tested the possibility of employing TRV vector to silence genes in Chinese white poplar(P.tomentosa Carr.) and wood-oil trees including tung tree(Vernicia fordii)and tea-oil tree(Camellia oleifera)with the marker gene PDS. Using TRV-based VIGS vectors,we also reported silencing of endogenous PDS and cloroplastos alterados1gene (CLA1)in tung tree with heterologous gene sequences from poplar,respectively.The loss-of-function assay based on heterologous VIGS developed in this study provides an alternative tool for functional genomics studies of woody plant species.

Materials and methods

Plant material and growth conditions

Seeds of tobacco(Nicotiana benthamiana L.)were ger-minated at25°C and seedlings were grown in plastic pots (10cm diameter)containing potting mixture.Plants were grown in growth rooms at22–25°C with60%relative humidity and a12h light:12h dark photoperiod cycle with light intensity ranging from300to400l mol m-2s-1.

Seeds of tung tree(Vernicia fordii)and tea-oil tree (Camellia oleifera)were stored at4°C and germinated seeds were sown in soil and grown in a growth chamber (25°C,16h:8h light:dark photoperiod).

Adventitious shoots of Chinese white poplar(Populus tomentosa Carr.)clone741were regenerated in woody plant medium(WPM)(Lloyd and McCown1980).Rooted plantlets were transferred to potting mix and grown in the greenhouse.Two-month-old seedlings were used for fur-ther experiments.

Cloning of PDS and CLA1genes

Total RNA was isolated from leaves of woody plants using RN09-EASYspin RNA Plant Mini Kit(Aidlab Biotech-nologies Co.,Ltd,Beijing,China),following the manu-facturer’s instructions.First-strand cDNA was synthesized from2l g DNase-treated RNA with RT-AMV transcrip-tase(TaKaRa,Dalian,China)in a total volume of20l l using oligo d(T)at42°C for30min.In order to get the accurate PDS fragments from V.fordii and C.oleifera, gene-speci?c primers were designed based on ESTs of PDS from Jatropha curcas(Ye et al.2009)and Camellia sin-ensis,respectively.The genome of poplar has been com-pleted(Tuskan et al.2006),therefore,sequences of PtoPDS and PtoCLA1were used as the templates to design the primers.The fragments of the PDS genes from different plants were ampli?ed by Taq DNA polymerase(TakaRa, Dalian,China),respectively.Positive clones of every fragment were con?rmed by DNA sequencing(BGI, Shenzhen,China).All PDS and PtoCLA1gene sequences were deposited in GenBank with the accession numbers and primer sequence information and fragment sizes of the PCR products used in this study are listed in Supplemen-tary Material Table S1.

Sequence alignment and identity calculation

The alignments of the nucleotide sequences of PDS and CLA1were performed using Clustal X1.81(http://www. https://www.sodocs.net/doc/185449689.html,/).The parameters of alignment are used as follows:gap opening penalty,10.00(both in pairwise alignment and multiple alignment);gap extension penalty, 0.20(both in pairwise alignment and multiple alignment); protein weight matrix,gonnet;residue-speci?c penalties, ON;hydrophilic penalties,ON;gap separation distance,0; end gap separation,ON;use negative matrix,OFF;and delay divergent cutoff(%),30.Identity calculation between sequences was performed using DNAman soft-ware(https://www.sodocs.net/doc/185449689.html,/).

Construction of TRV plasmids

The DNA fragments of VfPDS,CoPDS and PtoPDS were ampli?ed using a pair of primers containing Xho I and Sac I. NbPDS and PtoCLA1fragments were generated by RT-PCR with primers containing Xho I and Eco RI restriction sites.The pTRV1and pTRV2vectors described by Liu et al.(2002)were used in this study.The DNA products were double-digested and ligated into pTRV2with the same enzymes,respectively.The resulting vectors were designated as pTRV2-VfPDS(PDS from V.fordii), pTRV2-CoPDS(PDS from C.oleifera),pTRV2-PtoPDS (PDS from P.tomentosa Carr.),pTRV2-NbPDS(PDS from N.benthamiana)and pTRV2-PtoCLA1(CLA1from P. tomentosa Carr.).Plasmids were introduced into Agro-bacterium tumefaciens strain GV3101by freezing-thawing method(Ho¨fgen and Willmitzer1988).

Agrobacterium in?ltration

For the VIGS assay,a5-mL culture of Agrobacterium strain GV3101containing pTRV1and pTRV2was grown

Plant Cell Tiss Organ Cult

overnight at28°C in Luria-Broth(LB)medium containing three antibiotics(40mg L-1rifampicin,50mg L-1gen-tamycin and50mg L-1kanamycin).The next day,500l L of each Agrobacterium culture was inoculated into a 50mL LB medium containing antibiotics,10mM MES(2-(4-Morpholino)-Ethane Sulfonic Acid)and20l M aceto-syringone.The culture was grown overnight in a28°C shaker at the speed of200r min-1.Agrobacterium cells were harvested and resuspended in in?ltration buffer (10mM MgCl2,10mM MES,200l M acetosyringone,pH 5.6)to a?nal O.D.600of 1.0.Agrobacterium cultures containing pTRV1and pTRV2or its derivatives were mixed at1:1ratio and incubated for3h at room temper-ature.The syringe method operated the same as described previously(Burton et al.2000;Liu et al.2002). Effectiveness of gene silencing

The effectiveness of gene silencing,in N.benthamiana that showed photobleaching symptoms,was calculated by com-paring the number of leaves that showed symptoms with the total number of leaves on the plant.The formulas and method are described previously(Senthil-Kumar et al.2007). RT-PCR analysis

To detect the presence of TRV virus,RNA1and RNA2of TRV were ampli?ed by two pairs of speci?c primers as follows:TRV1-forward primer:50-GTAGGAGGAAGA GACCGAAG-30,TRV1-reverse primer:50-TAGTCGAAT CAGTAGCAACC-30,TRV2-forward primer:50-GTATGT CAGTGATCGCAGTAG-30,TRV2-reverse primer:50-CGT CCGTTTAGACGCTTGCGTAGG-30.To quantify PDS and CLA1transcript abundances in TRV2::PDS(PDS from different plant species)inoculated plants,semi-quantitative RT-PCR was performed as described before(Burton et al. 2000;Liu et al.2002).Primers that annealed outside the region targeted for silencing were used to ensure that only the endogenous gene would be tested and excluded the inter-ference of the sequences carried on the TRV2derivatives. The18S gene was used as an internal control for RNA quantity in semi-quantitative RT-PCR.PCR products were separated on a1%TAE gel and visualized by EtBr staining. At least three silencing plants for each construct were chosen for RT-PCR.Three technical replicates were performed for each sample.All the primers used for RT-PCR are shown in Supplementary Material Table S2.

Extraction and measurement of chlorophyll

Total chlorophyll was extracted from200mg of leaf tissue in an acetone:dimethyl sulphoxide(DMSO)(1:1v/v)mix.The supernatant was made up to1mL using this mix.The absorbance was recorded at663and645nm using UV–visible spectrophotometer Model DU800(Shimadzu Cor-poration,Kyoto,Japan).Total chlorophyll was measured as described previously(Hiscox and Israelstam1979)and expressed as the percentage reduction relative to the cor-responding control(wild-type or mock-in?ltrated).Three silencing plants were examined and all tests were con-ducted by performing three technical replicates for each sample in the assay.

Statistical analysis

The Student’s t test program(https://www.sodocs.net/doc/185449689.html,/ quickcalcs/ttest1.cfm)was used for statistical analysis of the data in the experiments of quantitative RT-PCR and measurement of chlorophyll.In all these experiments,it was found that the quantitative differences between the two groups of data for comparison were statistically signi?cant (P\0.05).

Results

TRV-mediated VIGS in V.fordii

In order to investigate whether the TRV vector can directly infect tung tree,a mixture of Agrobacterium cultures containing pTRV1and pTRV2constructs in a1:1ratio was in?ltrated into two or three leaves of three-week-old tung tree.The seedlings infected were grown at25°C.Four weeks after agroin?ltration,more than twenty plants inoculated with TRV1and TRV2vectors showed no obvious differences in overall shoot and leaf morphology compared with the control(Fig.1a).

To assess the infection and spread of TRV virus,total RNA was isolated from the newly developed true leaves of these in?ltrated plants.RT-PCR with TRV1-RNA1-spe-ci?c and TRV2-RNA2-speci?c primers showed that TRV virus was detected in all tested seedlings,while no speci?c product was detected in control plants(Fig.1b),indicating that tung tree could be infected by TRV virus.These results suggested that TRV-based VIGS vector could be used for silencing genes in tung tree.

Silencing of the PDS gene from V.fordii

by Agrobacterium-mediated VIGS

The phytoene desaturase(PDS)gene,which is highly conserved in various plant species(Liu et al.2002;Senthil-Kumar et al.2007),has been widely used as a VIGS marker in various plant species(Ratcliff et al.2001;Liu et al.2002;Turnage et al.2002).The silencing of PDS

Plant Cell Tiss Organ Cult

results in white leaves caused by photobleaching,which occurs in the absence of the gene product.In a previous study,Ye et al.(2009)successfully developed high throughput screening of gene function by TRV-based VIGS in Jatropha curcas ,a woody oil plant belonging to the Euphorbiaceae family.To determine if endogenous gene silencing can also be elicited by TRV-mediated VIGS in other Euphorbiaceae species,we ampli?ed a 261-bp fragment of VfPDS from V.fordii by a standard homology-based cloning technique.Sequencing results revealed that the partial coding sequence of VfPDS cloned shares 83.14and 83.52%nucleotide sequence identities with those of Arabidopsis thalianan and Nicotiana benthamiana ,respectively (Supplementary Material Fig.S1).The frag-ment of VfPDS was inserted into a pTRV-RNA2VIGS vector,pYL156(Liu et al.2002).The TRV vectors were delivered to the leaf cells of young tung tree plants by a simple agrobacterium in?ltration.All of these plants in?l-trated with pTRV2-VfPDS exhibited photo-bleaching symptoms on the upper leaves 25days post agroin?ltration and the symptoms continued to appear in the leaves which subsequently developed one month later (Fig.2a).In addition,the bleaching seems to be more in the phloem or in the veins which may be involved in vascular movement

of plant virus (Se

′ron and Haenni 1996).While plants infected with the pTRV2empty vector did not display any photobleaching phenotype and grew normally (Fig.2a).To monitor the silencing level of VfPDS ,we performed semi-quantitative RT-PCR with speci?c primers of pTRV1and pTRV2.The result revealed that the PDS transcript

levels in photobleaching leaves were reduced by more than 80%compared to the controls infected with TRV alone (Fig.2b).

TRV-mediated VIGS in Populus and C.oleifera In poplar,a candidate for development of a VIGS vector is poplar mosaic virus (PopMV ),which is a RNA carlavirus that naturally infects species and hybrids in the genus Populus .Recently,Naylor et al.(2005)demonstrated that a VIGS vector based on the genome sequence of PopMV (Smith and Campbell 2004)was successfully applied in N.benthamiana for suppression of a GFP reporter gene.However,it is not clear whether this virus vector can suppress effectively the expression of endogenous genes in poplar so far.To test the infection ability of TRV in poplar,PtoPDS was isolated from Chinese white poplar (P.tom-entosa Carr.)based on homology (Supplementary Material Fig.S1).Three weeks after agroin?ltration,approximately 30%of poplar plants treated with pTRV2-PtoPDS developed photobleaching symptoms on the upper newly-grown leaves (Fig.3a),but silencing symptoms rapidly disappeared on the newly formed leaves one month post inoculation (Fig.3b).Plants in?ltrated with the pTRV2empty vector did not exhibit any photobleaching phenotype and grew normally (Fig.3a).Endogenous transcript levels of PtoPDS were assessed by RT-PCR.The results showed that a slight decrease in PDS transcript levels was detected by 20days post in?ltration in these plants in?ltrated with pTRV2-PtoPDS .But transcript suppression was recovered partially after 40days (data not shown).These

data

Fig.1Agroinfection of tung tree plant with tobacco rattle virus (TRV)vector.a Tung tree plant in?ltrated with mixed Agrobacterium cultures containing pTRV1and pTRV2(right )and untreated plant (left ).b Viral transcripts (TRV1and TRV2)in leaves by Reverse Transcriptase (RT)-PCR.Six mock-treated plants were determined and all of them contained TRV transcripts.rRNAs was used as internal

controls

Fig.2Silencing of endogenous phytoene desaturase (PDS )gene in seedlings of tung tree.a Control and TRV-VfPDS -treated plants 25days after in?ltration.Photobleached phenotype was observed in the plant (right )infected with TRV-VfPDS while the plant treated with TRV alone (left )remained green.b RT-PCR analysis in PDS silenced plant.PCRs with cycles of 20,23,26,29and 32

Plant Cell Tiss Organ Cult

indicated that TRV-based VIGS is not effective to silence endogenous genes in Populus .

Tea oil tree (C.oleifera )is mainly cultivated in trop-ical and subtropical climates and its seeds can be pressed to yield edible oil.To determine whether the TRV-vector ef?ciently mediates gene silencing in tea oil tree,we cloned a 366-bp fragment of CoPDS by PCR ampli?ca-tion based on the homologous sequence from Camellia sinensis.Sequence comparison of CoPDS and its homo-logs in J.curcas ,Populus ,tobacco and tomato revealed a nucleotide acid identity level of 85–93%(Supplementary Material Fig.S1).We used the Agrobacterium -in?ltration method mentioned above to induce VIGS.The photo-bleaching phenotype of PDS was not visible on upper leaves of treated plants even 2months after infection (Supplementary Material Fig.S2A).Furthermore,we did not detect TRV1or TRV2transcripts in all treated plants (Supplementary Material Fig.S2B).This result suggested that tea-oil tree could not be infected by TRV-based vectors.

Silencing of the PDS gene in N.benthamiana using heterologous PDS gene sequences from woody plant species

It has been well established that TRV-mediated VIGS is highly ef?cient in N.benthamiana (Lu et al.2003;Burch-Smith et al.2004).Senthil-Kumar et al.(2007)demon-strated that heterologous PDS gene sequences from a wide range of plant species could be applied to suppress the expression of their orthologs in N.benthamiana .To test whether heterologous gene sequences from woody plant species can be used to silence their respective orthologs in N.benthamiana ,PDS fragments from P.tomentosa Carr.,C.oleifera and V.fordii were inserted into the TRV2vector and used to perform VIGS in N.benthamiana ,respectively.As controls,PDS sequences from N.benthamiana and tomato (S.lycopersicum )were also been cloned into the TRV2vector.We in?ltrated tobacco leaves with the TRV-vectors containing these PDS genes from various plant species,respectively.A visible bleached phenotype was observed in all treated plants but the extent of photoble-aching varied from mild to almost complete bleaching among the different PDS sequences used for gene silencing (Fig.4a).

We investigated the frequency of gene silencing in N.benthamiana when infected with different PDS sequences.The gene silencing effectiveness was calculated by com-paring the number of photobleaching leaves with the total number of leaves inoculated with TRV2::PDS .A wide range of variation in silencing effectiveness was found among the plant species (Fig.4b).N.benthamiana showed the highest effectiveness (95%)of silencing and SlPDS sequences caused photobleaching in approximately 90%of the plants tested. C.oleifera and V.fordii showed silencing frequencies of only 51and 63%,respectively.The effectiveness of heterologous PDS silencing was the lowest (46%)in Populus (Fig.4b).These results sug-gested that the effectiveness of gene silencing was rela-tively low in N.benthamiana when heterologous gene sequences from woody plant species,especially for Popu-lus ,were used for VIGS.

Furthermore,we determined the ef?ciency of gene silencing triggered by heterologous PDS gene sequences in N.benthamaina .qRT-PCR analysis showed that a marked reduction in gene expression occurred in these plants infected with TRV2-NbPDS and TRV2-SlPDS ,but only a reduction of twofold in plants infected with TRV2-PtoPDS compared with the control (Fig.4c).Chlorophyll was extracted from both green control leaves and the whole photobleaching leaves.Plants infected with TRV2-NbPDS showed almost 100%reductions of total chlo-rophyll compared with wild-type plants.The content of chlorophyll was reduced by 87%in the TRV2-

SlPDS

Fig.3Silencing of PDS in seedlings of Populus .a The control plant infected with mixed Agrobacterium cultures containing pTRV1and pTRV2remained green 3weeks after in?ltration while photobleached phenotype (shown with red arrow )was observed in the plant in?ltrated with TRV-PtoPDS whose phenomenon was not so strong.PtoPDS expression was analyzed by RT-PCR in silenced plant.PCRs with cycles of 22,24,26,28and 30were performed by PtoPDS -speci?c primers.b Photobleached phenotype was observed in the old leaves but not the newly ones (shown with red arrows ).TRV1transcripts were visible in leaves by RT-PCR.1-G:green newly leaves,1-W:old leaves with photobleached phenotype.(Color ?gure online)

Plant Cell Tiss Organ Cult

silenced plants.While plants inoculated with the PtoPDS ,CoPDS and VfPDS clones showed reductions of less than 20,45and 60%in chlorophyll content,respectively (Fig.4d).These results clearly demonstrated that heter-ologous gene sequences from woody plants could not be used to effectively silence their respective orthologs in N.benthamiana .

Silencing of heterologous PDS gene in tung tree by CoPDS gene sequences

We have previously demonstrated that TRV virus could infect and spread rapidly in tung tree (Fig.1).To investi-gate whether heterologous virus-induced gene silencing is an effective tool in functional genomic analysis of

woody

Fig.4Silencing of the PDS gene in N.benthamiana using heterologous PDS gene

sequences from different plant species by TRV-mediated VIGS.Plants were in?ltrated with TRV1?TRV2-PDS (PDS from different species).a The photobleaching phenotype was photographed at 20days post in?ltration.b The effectiveness of gene silencing was calculated by counting the number of leaves that showed

photobleaching on a gene-silenced plant at 20days post in?ltration.c qRT-PCR analysis in PDS -silenced plant.d The quanti?cation of chlorophyll content in gene-silenced plants at 20days post in?ltration.The values are means of three independent experiments and the bars represent standard error.Statistical differences were determined using the Student’s t test;*P \0.05

Plant Cell Tiss Organ Cult

plants,in an initial experiment,we in?ltrated young leaves of three-week-old tung tree with CoPDS from tea-oil tree whose identity is 88.15%aligned with VfPDS .The ?rst effects of the silencing became visible approximately 20days after Agrobacterium inoculation as discoloration of

newly emerging leaves (Fig.5b).Five days later,some leaves showed almost complete discoloration (Fig.5c).In most of infected plants,CoPDS silencing remained easily detectable throughout subsequent developmental stages.The CoPDS silencing response did not seem to move below the node of leaf infection,many plants in?ltrated with TRV-VIGS vector showed complete discoloration of all newly emerging leaves.In some cases,the silencing response disappeared so that normal green leaves devel-oped again above photobleaching leaves (Fig.5d).While plants in?ltrated with the pTRV2empty vector grew nor-mally and did not exhibit any photobleaching phenotype (Fig.5a).These results indicated that heterologous gene sequences could effectively silence their respective ortho-logs by TRV-mediated VIGS in tung tree.

Silencing of VfPDS and VfCLA1by heterologous gene fragments from P.tomentosa Carr

To examine whether a heterologous gene sequence from a distantly related species can trigger VIGS in tung tree,we in?ltrated seedlings of tung tree with PtoPDS from P.tomentosa Carr.Photobleaching phenotype in the upper new leaves of tung tree was initially observed 20days after in?ltration with PtoPDS -expressing Agrobacteria,but the discoloration was limited to the main veins of newly formed leaves (Fig.6a),while the control plants infected with TRV vector alone showed normal growth (Fig.6a).These results indicated that the heterologous PDS gene sequence from Populus in a TRV-based VIGS vector was capable of suppressing expression of its ortholog in tung tree.

In plants,CLA1encodes 1-deoxyxylulose 5-phosphate synthase which is the ?rst enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway involved in chloroplast development (Estevez et al.2000).The CLA1gene is highly conserved in various plant species (Mandel et al.1996).Silencing of CLA1in Arabidopsis and S.melongena plants led to an albino phenotype on true leaves (Mandel et al.1996;Estevez et al.2000,2001).We obtained a 507bp fragment of the PtoCLA1gene from P.tomentosa Carr.by RT-PCR.Sequencing results showed that a poplar CLA1gene (PtoCLA1)shared 91.05%identity with

tung

Fig.5PDS silenced by TRV-CoPDS in seedling of tung tree.a Plant treated with TRV vector (left )remained green.b ,c and d shown the development of photobleached phenotype for 20,25and 30days after agro-in?ltration,

respectively

Fig.6Silencing of VfPDS and VfCLA1by a heterologous gene fragment from Populus tomentosa.a TRV-PtoPDS -and TRV-PtoCLA1-treated plants 20days after in?ltration.Photobleached phenotype was observed in the plant in?ltrated with TRV-PtoPDS and TRV-PtoCLA1while the control remained green.b RT-PCR analysis in PDS and CLA1silenced plant,respectively.PCRs with cycles of 22,24,26,28and 30were performed by PtoPDS -or PtoCLA1-speci?c primers.c Comparison of the content of chloro-phyll a,chlorophyll b and total chlorophyll between silenced and control plants.Error bars represent SE of three replications.Statistical differences were determined using the Student’s t test;*P \0.05

Plant Cell Tiss Organ Cult

tree VfCLA1at the nucleotide level(Supplementary Material Fig.S3).The PtoCLA1fragment was inserted into TRV2and seedlings of tung tree were in?ltrated with Agrobacterium cultures containing the TRV1and TRV2 constructs.Photobleaching phenotype was visible in the upper new leaves of all treated plants about20days post in?ltration(Fig.6a).

In order to con?rm that observed photobleaching was due to silencing of the VfPDS and VfCLA1genes at the molecular level,we performed semi-quantitative RT-PCR on all treatment https://www.sodocs.net/doc/185449689.html,pared to the untreated plants, the target mRNAs were reduced to approximate75%in the targeted gene silenced plants(Fig.6b).PDS and CLA1 enzymes are involved in chlorophyll synthesis.The con-tents of the chlorophyll a,chlorophyll b and total chloro-phyll in PtoPDS-silenced plants were reduced by35% compared to that of the controls infected with TRV alone. Meanwhile,the content of the total chlorophyll in PtoCLA1 silenced plants was also decreased greatly compared to that of controls(Fig.6c).The results suggested that heterolo-gous VIGS could be ef?ciently implemented to silence PDS and CLA1in tung tree.

Discussion

Virus-induced gene silencing(VIGS)as one of the reverse genetic tools has been used routinely for analysis of gene function in many plant species(Baulcombe1999;Burch-Smith et al.2004),mainly because VIGS is a very simple and robust method that does not require stable transfor-mation which is a laborious,time-consuming and small-scale work.More importantly,this method can use heter-ologous gene sequences to silence orthologs in plants that do not have extensive gene sequence information.In this study,we developed a protocol for VIGS in tung tree(V. fordii)using a TRV vector.The TRV vectors carrying PDS gene fragment of tung tree induced a high-ef?ciency silencing by Agrobacterium-mediated VIGS(Fig.2).We also carried out a systematic study to determine the feasi-bility of using heterologous gene sequences from various wood species to silence their orthologs in N.benthamiana. As measured by suppression of target transcript levels,the VIGS ef?ciency of the PDS gene in silenced tobacco plants was46–63%(Fig.4b),suggesting that VIGS by TRV vector in tobacco was not effectively induced when using heterologous gene fragments from wood plant species.But we found that PtoPDS and PtoCLA1gene sequences from P.tomentosa Carr.as well as CoPDS sequence from C. oleifera could be used to silence their respective orthologs in tung tree(Figs.5,6).These data indicated that the TRV-based VIGS should have broad applications for functional genomics studies in heterologous wood species.

To date,there have been minimal effective methods for gene silencing in tree species except for the use of trans-genic procedures.However,successful transformation is con?ned to a limited species and cultivars in trees.It is also time-consuming and labor intensive due to low transfor-mation ef?ciency in most of woody plants.VIGS vector systems offer an alternative for characterization of gene functions without plant transformation(Ratcliff et al.2001; Holzberg et al.2002;Liu et al.2002;Gronlund et al.2008). In these VIGS systems previously reported,the most widely used VIGS vectors are derived from the tobacco rattle virus,which has been commonly used in many her-baceous plants(Burch-Smith et al.2004).The TRV invades a wide range of hosts and is able to spread vig-orously throughout the entire plant(Senthil-Kumar et al. 2007).Recently,a TRV-based VIGS assay was success-fully developed in cotton,which was a shrub native to tropical and subtropical regions around the world,and the genes of interest were readily silenced with an ef?ciency of 100%after inoculation at the seedling stage(Gao et al. 2011).Qu et al.(2012)further reported that the TRV-VIGS system could be used for rapid functional analysis of genes involved in cotton?ber development.In addition,it has also been well demonstrated that TRV vector could trigger VIGS in J.curcas,a small woody plant belonging to Eu-phorbiaceae(Ye et al.2009).Tung tree(V.fordii)is another species of the Euphorbiaceae family,native to southern China and northern Vietnam.It is valuable for tung oil,which sources from the oilseeds of the tree and commonly used in formulations of inks,dyes,coatings,and resins.A recent study has demonstrated that tung oil is a raw material for biodiesel production after blending with other biodiesel(Chen et al.2010).However,less is known about functions of these genes involved in oil biosynthetic pathway in tung seeds.In addition,there is no report of a method for genetic transformation of tung tree so far.VIGS is a rapid,simple and robust method for determining and studying the function of plant genes and does not require stable transformation.In this paper,we demonstrated for the?rst time that TRV system could effectively induce endogenous gene silencing in seedlings of tung tree (Fig.2).Therefore,TRV-mediated VIGS provides a con-venient and effective method to achieve functional gene data in tung tree.

The rapid innovation of sequencing technologies is vastly expanding the sequence database for important plant species,the majority of which are dif?cult to manipulate for functional genomics studies.For example,Populus is one of favorable model tree because of its small genome and its genome sequencing has been completed and released in2006(Tuskan et al.2006).Therefore,the study on functional genomics in Populus has become a major task.Agrobacterium-mediated genetic transformation is a

Plant Cell Tiss Organ Cult

powerful tool and an ef?cient technique for the study of plant functional genomics,however,it is both time-con-suming and labour-intensive.VIGS provides a rapid and powerful tool to dissect gene functions in species that are not amenable to stable genetic https://www.sodocs.net/doc/185449689.html,rge-scale VIGS experiments have been adopted as a fast-forward genetics approach to screen for interesting phenotypes (Baulcombe1999;Burch-Smith et al.2004).More importantly,this technique can use heterologous gene sequences to silence orthologs in plants(Senthil-Kumar et al.2007).But reliability and effectiveness of VIGS systems depends on both plant species and virus vectors.In this study,we demonstrated that the VIGS system using TRV vector only induced moderate reduction of PtoPDS-mRNA levels in Populus(Fig.3),and even C.oleifera could not be infected by TRV vector(Supplementary material Fig.S2).Fortunately,the TRV-VIGS system was shown to be effective in tung tree,when a marker gene VfPDS was silenced,resulting in a photobleaching phe-notype(Fig.2).In order to develop a high throughput loss-of-function assay for functional genomic studies in Popu-lus,we successfully silenced PDS and CLA1in tung tree using heterologous gene sequences from P.tomentosa Carr.(Fig.6).Based on these results,we believe that the TRV-mediated VIGS could be applied broadly for func-tional genomics studies in tung tree using heterologous gene sequences from Populus,indicating that heterologous VIGS is a promising approach in functional genomics of woody plants.

In conclusion,we have demonstrated that TRV-medi-ated VIGS can be used in a wide range of Euphorbiaceae species,and this adds to the increasing list of wood species that are able to be used for VIGS-mediated studies.We have also developed the TRV-VIGS system to silence effectively their respective orthologs in tung tree using heterologous gene sequences from even distantly related species such as Populus and C.oleifera.The lose-of-function assay by TRV-mediated VIGS developed in this study provides an alternative tool for functional genomics studies of Populus genes.

Acknowledgments This work was supported by the National Natural Science Foundation of China(31171620,31370672),the National Key Project for Research on Transgenic Plant(2011ZX08010-003),the program for New Century Excellent Talents in University(NCET-11-0700)and The Research Fund for the Dectoral Program of Higher Education(20110182110004).

References

Baulcombe D(1999)Viruses and gene silencing in plants.Arch Virol Suppl15:189–201

Becker A,Lange M(2010)VIGS–genomics goes functional.Trends Plant Sci15(1):1–4Benedito VA,Visser PB,Angenent GC,Krens FA(2004)The potential of virus-induced gene silencing for speeding up functional characterization of plant genes.Genet Mol Res 3(3):323–341

Borevitz JO,Ecker JR(2004)Plant genomics:the third wave.Annu Rev Genomics Hum Genet5:443–477

Brigneti G,Martin-Hernandez AM,Jin H,Chen J,Baulcombe DC, Baker B,Jones JD(2004)Virus-induced gene silencing in Solanum species.Plant J39(2):264–272

Burch-Smith TM,Anderson JC,Martin GB,Dinesh-Kumar SP(2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants.Plant J39(5):734–746

Burton RA,Gibeaut DM,Bacic A,Findlay K,Roberts K,Hamilton A,Baulcombe DC,Fincher GB(2000)Virus-induced silencing of a plant cellulose synthase gene.Plant Cell12(5):691–706 Candela H,Perez-Perez JM,Micol JL(2011)Uncovering the post-embryonic functions of gametophytic-and embryonic-lethal genes.Trends Plant Sci16(6):336–345

Chen YH,Chen JH,Chang CY,Chang CC(2010)Biodiesel production from tung(Vernicia montana)oil and its blending properties in different fatty acid compositions.Bioresour Tech-nol101(24):9521–9526

Chung E,Seong E,Kim YC,Chung EJ,Oh SK,Lee S,Park JM, Joung YH,Choi D(2004)A method of high frequency virus-induced gene silencing in chili pepper(Capsicum annuum L.cv.

Bukang).Mol Cells17(2):377–380

Constantin GD,Krath BN,MacFarlane SA,Nicolaisen M,Johansen IE,Lund OS(2004)Virus-induced gene silencing as a tool for functional genomics in a legume species.Plant J40(4): 622–631

Dinesh-Kumar SP,Anandalakshmi R,Marathe R,Schiff M,Liu Y (2003)Virus-induced gene silencing.Methods Mol Biol 236:287–294

Ding XS,Schneider WL,Chaluvadi SR,Mian MA,Nelson RS(2006) Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.Mol Plant Microbe Interact19(2):1229–1239

Doja VV,Koonin EV(2013)The closterovirus-derived gene expres-sion and RNA interference vectors as tools for research and plant biotechnology.Front Microbiol4:83

Ekengren SK,Liu Y,Schiff M,Dinesh-Kumar SP,Martin GB(2003) Two MAPK cascades,NPR1,and TGA transcription factors play

a role in Pto-mediated disease resistance in tomato.Plant J

36(6):905–917

Estevez JM,Cantero A,Romero C,Kawaide H,Jimenez LF, Kuzuyama T,Seto H,Kamiya Y,Leon P(2000)Analysis of the expression of CLA1,a gene that encodes the1-deoxyxylulose 5-phosphate synthase of the2-C-methyl-D-erythritol-4-phos-phate pathway in Arabidopsis.Plant Physiol124(1):95–104 Estevez JM,Cantero A,Reindl A,Reichler S,Leon P(2001) 1-Deoxy-D-xylulose-5-phosphate synthase,a limiting enzyme for plastidic isoprenoid biosynthesis in plants.J Biol Chem 276(25):22901–22909

Faivre-Rampant O,Gilroy EM,Hrubikova K,Hein I,Millam S, Loake GJ,Birch P,Taylor M,Lacomme C(2004)Potato virus X-induced gene silencing in leaves and tubers of potato.Plant Physiol134(4):1308–1316

Fofana IB,Sangare A,Collier R,Taylor C,Fauquet CM(2004)A geminivirus-induced gene silencing system for gene function validation in cassava.Plant Mol Biol56(4):613–624

Gao X,Wheeler T,Li Z,Kenerley CM,He P,Shan L(2011) Silencing GhNDR1and GhMKK2compromises cotton resis-tance to Verticillium wilt.Plant J66(2):293–305

Gronlund M,Constantin G,Piednoir E,Kovacev J,Johansen IE,Lund OS(2008)Virus-induced gene silencing in Medicago truncatula and Lathyrus odorata.Virus Res135(2):345–349

Plant Cell Tiss Organ Cult

Hamilton AJ,Baulcombe DC(1999)A species of small antisense RNA in posttranscriptional gene silencing in plants.Science 286(5441):950–952

Hiscox JD,Israelstam GF(1979)a method for the extraction of chlorophyll from leaf tissue without maceration.Can J Bot 57:1332–1334

Ho¨fgen R,Willmitzer L(1988)Storage of competent cells for Agrobacterium transformation.Nucl Acids Res16:9877 Holzberg S,Brosio P,Gross C,Pogue GP(2002)Barley stripe mosaic virus-induced gene silencing in a monocot plant.Plant J 30(3):315–327

Howes T,Kumagai MH(2005)Virus-induced gene silencing in Nicotiana benthamiana using DEAD-box helices sequence derived from Dunaliella salina.J Young Investig12:1–6 Kurth EG,Peremyslov VV,Prokhnevsky AI,Kasschau KD,Miller M,Carrington JC,Dolja VV(2012)Virus-derived gene expres-sion and RNA interference vector for grapevine.J Virol 86(11):6002–6009

Lansac M,Eyquard JP,Salvador B,Garcia JA,Le Gall O,Decroocq V,Schurdi-Levraud EV(2005)Application of GFP-tagged Plum pox virus to study Prunus-PPV interactions at the whole plant and cellular levels.J Virol Methods129(2):125–133

Liu Y,Schiff M,Dinesh-Kumar SP(2002)Virus-induced gene silencing in tomato.Plant J31(6):777–786

Lloyd G,McCown B(1980)Commercially feasible micropropagation of mountain laurel(Kalmla latlfolia)by use of shoot tip cultures.

Comb Proc Int Plant Prop Soc30:421–427

Lu R,Martin-Hernandez AM,Peart JR,Malcuit I,Baulcombe DC (2003)Virus-induced gene silencing in plants.Methods 30(4):296–303

Mandel MA,Feldmann KA,Herrera-Estrella L,Rocha-Sosa M,Leon P(1996)CLA1,a novel gene required for chloroplast develop-ment,is highly conserved in evolution.Plant J9(5):649–658 Martienssen RA(1998)Functional genomics:probing plant gene function and expression with transposons.Proc Natl Acad Sci U S A95(5):2021–2026

Naylor M,Reeves J,Cooper JI,Edwards ML,Wang H(2005) Construction and properties of a gene-silencing vector based on Poplar mosaic virus(genus Carlavirus).J Virol Methods 124(1–2):27–36

Ostergaard L,Yanofsky MF(2004)Establishing gene function by mutagenesis in Arabidopsis thaliana.Plant J39(5):682–696 Pagliarani G,Paris R,Iorio AR,Tartarini S,Del Duca S,Arens P, Peters S,van de Weg E(2012)Genomic organisation of the Mal d1gene cluster on linkage group16in apple.Mol Breed 29(3):759–778

Qu J,Ye J,Geng YF,Sun YW,Gao SQ,Zhang BP,Chen W,Chua NH(2012)Dissecting functions of KATANIN and WRIN-KLED1in cotton?ber development by virus-induced gene silencing.Plant Physiol160(2):738–748

Ratcliff F,Martin-Hernandez AM,Baulcombe DC(2001)Technical advance.Tobacco rattle virus as a vector for analysis of gene function by silencing.Plant J25(2):237–245

Robertson D(2004)VIGS vectors for gene silencing:many targets, many tools.Annu Rev Plant Biol55:495–519

Robinson SJ,Parkin IA(2009)Bridging the gene-to-function knowledge gap through functional genomics.Methods Mol Biol 513:153–173

Ryu CM,Anand A,Kang L,Mysore KS(2004)Agrodrench:a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species.Plant J 40(2):322–331

Sasaki S,Yamagishi N,Yoshikawa N(2011)Ef?cient virus-induced gene silencing in apple,pear and Japanese pear using Apple latent spherical virus vectors.Plant Methods7(1):15Scalabrin S,Troggio M,Moroldo M,Pindo M,Felice N,Coppola G, Prete G,Malacarne G,Marconi R,Faes G,Jurman I,Grando S, Jesse T,Segala C,Valle G,Policriti A,Fontana P,Morgante M, Velasco R(2010)Physical mapping in highly heterozygous genomes:a physical contig map of the Pinot Noir grapevine cultivar.BMC Genomics11:204

Senthil-Kumar M,Udayakumar M(2006)High-throughput virus-induced gene-silencing approach to assess the functional relevance of a moisture stress-induced cDNA homologous to lea4.J Exp Bot57(10):2291–2302

Senthil-Kumar M,Hema R,Anand A,Kang L,Udayakumar M, Mysore KS(2007)A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing.New Phytol176(4):782–791

Se′ron K,Haenni A-L(1996)Vascular movement of plant viruses.

Mol Plant Microbe Interact9(6):435–442

Smith CM,Campbell MM(2004)Complete nucleotide sequence of the genomic RNA of poplar mosaic virus(Genus Carlavirus).

Arch Virol149(9):1831–1841

Turnage MA,Muangsan N,Peele CG,Robertson D(2002)Gemini-virus-based vectors for gene silencing in Arabidopsis.Plant J 30(1):107–114

Tuskan GA,Difazio S,Jansson S,Bohlmann J,Grigoriev I,Hellsten U,Putnam N,Ralph S,Rombauts S,Salamov A,Schein J,Sterck L,Aerts A,Bhalerao RR,Bhalerao RP,Blaudez D,Boerjan W, Brun A,Brunner A,Busov V,Campbell M,Carlson J,Chalot M, Chapman J,Chen GL,Cooper D,Coutinho PM,Couturier J, Covert S,Cronk Q,Cunningham R,Davis J,Degroeve S, Dejardin A,Depamphilis C,Detter J,Dirks B,Dubchak I, Duplessis S,Ehlting J,Ellis B,Gendler K,Goodstein D, Gribskov M,Grimwood J,Groover A,Gunter L,Hamberger B, Heinze B,Helariutta Y,Henrissat B,Holligan D,Holt R,Huang W,Islam-Faridi N,Jones S,Jones-Rhoades M,Jorgensen R, Joshi C,Kangasjarvi J,Karlsson J,Kelleher C,Kirkpatrick R, Kirst M,Kohler A,Kalluri U,Larimer F,Leebens-Mack J,Leple JC,Locascio P,Lou Y,Lucas S,Martin F,Montanini B,Napoli C,Nelson DR,Nelson C,Nieminen K,Nilsson O,Pereda V, Peter G,Philippe R,Pilate G,Poliakov A,Razumovskaya J, Richardson P,Rinaldi C,Ritland K,Rouze P,Ryaboy D, Schmutz J,Schrader J,Segerman B,Shin H,Siddiqui A,Sterky F,Terry A,Tsai CJ,Uberbacher E,Unneberg P,Vahala J,Wall K,Wessler S,Yang G,Yin T,Douglas C,Marra M,Sandberg G, Van de Peer Y,Rokhsar D(2006)The genome of black cottonwood,Populus trichocarpa(Torr.&Gray).Science 313(5793):1596–1604

Wang C,Cai X,Wang X,Zheng Z(2006)Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis.Funct Plant Biol33:347–355

Waterhouse PM,Graham MW,Wang MB(1998)Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc Natl Acad Sci U S A95(23):13959–13964

Wu J,Wang Z,Shi Z,Zhang S,Ming R,Zhu S,Khan MA,Tao S, Korban SS,Wang H,Chen NJ,Nishio T,Xu X,Cong L,Qi K, Huang X,Wang Y,Zhao X,Deng C,Gou C,Zhou W,Yin H, Qin G,Sha Y,Tao Y,Chen H,Yang Y,Song Y,Zhan D,Wang J,Li L,Dai M,Gu C,Shi D,Wang X,Zhang H,Zeng L,Zheng D,Wang C,Chen M,Wang G,Xie L,Sovero V,Sha S,Huang W,Zhang M,Sun J,Xu L,Li Y,Liu X,Li Q,Shen J,Paull RE, Bennetzen JL(2013)The genome of the pear(Pyrus bretsch-neideri Rehd.).Genome Res23(2):396–408

Ye J,Qu J,Bui HT,Chua NH(2009)Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencing.Plant Biotechnol J7(9):964–976

Plant Cell Tiss Organ Cult

Yu Q,Tong E,Skelton RL,Bowers JE,Jones MR,Murray JE,Hou S, Guan P,Acob RA,Luo MC,Moore PH,Alam M,Paterson AH, Ming R(2009)A physical map of the papaya genome with integrated genetic map and genome sequence.BMC Genomics 10:371Zhang C,Ghabrial SA(2006)Development of Bean pod mottle virus-based vectors for stable protein)expression and sequence-speci?c virus-induced gene silencing in soybean.Virology344(2): 401–411

Plant Cell Tiss Organ Cult

肺结核培训材料

结核病防治知识培训材料 一、什么是结核病? 结核病是由结核杆菌侵入人体后引起的一种慢性传染性疾病，长期以来因为没有有效药物治疗并具有较强的传染性，故人们对结核病产生了很强的恐惧心理. 随着科学技术的进展，科学工作者自50 年代以来，已经研制出十数种有效的抗结核药物，只要早期发现，正规治疗，是完全可以治愈的. 结核杆菌可以侵入人体任何器官，也就是说人体的各种器官都可以发生结核病，但结核杆菌主要通过人体的呼吸道进行传播，所以在人体感染结核杆菌后发生肺结核者占绝大多数. 因此，要控制乃至消灭结核病，加强对肺结核患者的治疗与管理，是当前结核病防治工作中的重点。 二、结核病是怎样传播的? 排菌的肺结核患者(痰里查到结核杆菌) ，是传播结核病的主要传染源. 当患者咳嗽、打喷囔或高声谈笑时，含结核菌的飞沫核从呼吸道直接排出，被健康人吸入后即形成结核感染，这是结核病传播的主要途径. 一个排菌的肺结核病人，尤其是痰液直接涂片检查阳性的排菌病人，一次咳嗽可喷出含有结核抨菌的微粒约3500 个，平常呼气时所呼出的带菌微粒则很少，大声说话一分钟约喷出微粒600-700 个，而打一次喷嚏播散到空气中的微粒竟高达100 万个. 可见排菌阳性的肺结核病人咳嗽，打喷嚏时的传里染危险性是相当严重的. 如果住室

经常开窗通风换气，就可大大减少传染的机会. 活动性肺结核病人，一般都有咳嗽，凡咳嗽两周以上者，均应常规进行X 线胸透和查痰，以早期发现肺结核病人，减少结核病的传播. 要加强对病人的教育，在咳嗽、打喷嚏时要用手帕或口罩作掩护，以免播散细菌，传给他人。 三、肺结核有那些主要症状? 肺结核早期或轻度肺结核，可无任何症状或症状轻微而被忽视，若病变处于活动进展阶段时，可出现以下症状: 1. 发热: 表现为午后低热，多在下午4-8 时体温升高，一般为37-38℃之间. 这时病人常常伴有全身乏力或消瘦，夜间盗汗，女性可导致月经不调或停经. 2. 咳嗽咳痰: 是肺结核最常见的早期症状，但也最易使患者或医生误以为是“感冒”或“气管炎’而导致误诊. 3. 痰中带血: 痰内带血丝或小血块，大多数痰内带血是由结核引起的. 四、怀疑自己有了肺结核怎么办? 当你明白了结核病是怎么一回事，知道了肺结核都有些什么症状，那么如果你具有前述症状而怀疑自己患肺结核时，特别是咳嗽咳痰，痰中带血已经超过两周以上，你就应立即去你所在地的结核病防治机构就诊，及早诊断，规则治疗，早日痊愈. 另外，排菌肺结核的亲属〈密切接触者〉，也应该及时进行健康查体。 五、怎样向医生叙述病史?

植物基因启动子的克隆方法及其应用

分子植物育种，２００６年，第４卷，第３（Ｓ）期，第８５－９１页 ＭｏｌｅｃｕｌａｒＰｌａｎｔＢｒｅｅｄｉｎｇ，２００６，Ｖｏｌ．４，Ｎｏ．３（Ｓ），８５－９１ 专题报告 Ｒｅｖｉｅｗ 植物基因启动子的克隆方法及其应用 尹辉李丹张毅李秋莉﹡ 辽宁师范大学生命科学学院，大连，１１６０２９ ﹡通讯作者，ｌｉｑｉｕｌｉ＠ｄｌ．ｃｎ 摘要植物基因的表达调控已成为分子生物学研究热点，启动子是基因表达调控的重要顺式元件，启动子的克隆对于研究基因表达调控、构建基因工程载体、表达目的蛋白有着重要的意义。启动子克隆的方法很多，从常用的启动子陷阱技术筛选启动子到ＰＣＲ方法的应用，此后相继问世的一些基于ＰＣＲ的克隆启动子技术，如载体锚定ＰＣＲ、反向ＰＣＲ、接头ＰＣＲ、交错热不对称ＰＣＲ等，为克隆启动子提供了更可靠，更合理的方法。本文着重介绍了几种植物基因启动子的克隆方法，分析了它们的优缺点，并展望了今后的研究前景。 关键词启动子，启动子陷阱技术，反向ＰＣＲ，锚定ＰＣＲ，交错热不对称ＰＣＲ ＣｌｏｎｉｎｇＭｅｔｈｏｄｓｏｆＰｌａｎｔＧｅｎｅＰｒｏｍｏｔｅｒｓａｎｄＴｈｅｉｒＡｐｐｌｉｃａｔｉｏｎｓ ＹｉｎＨｕｉＬｉＤａｎＺｈａｎｇＹｉＬｉＱｉｕｌｉ＊ ＣｏｌｌｅｇｅｏｆＬｉｆｅＳｃｉｅｎｃｅｓ，ＬｉａｏｎｉｎｇＮｏｒｍａｌＵｎｉｖｅｒｓｉｔｙ，Ｄａｌｉａｎ，１１６０２９ ﹡Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ，ｌｉｑｉｕｌｉ＠ｄｌ．ｃｎ ＡｂｓｔｒａｃｔＴｈｅｅｘｐｒｅｓｓｉｏｎａｎｄｒｅｇｕｌａｔｉｏｎｏｆｐｌａｎｔｇｅｎｅｈａｓｂｅｅｎｔｈｅｈｏｔｓｐｏｔｓｔｕｄｙｉｎｍｏｌｅｃｕｌａｒｂｉｏｌｏｇｙ．Ｐｒｏｍｏｔｅｒｉｓａｎｉｍｐｏｒｔａｎｔｃｉｓ－ａｃｔｉｎｇｅｌｅｍｅｎｔ．Ｃｌｏｎｉｎｇｐｒｏｍｏｔｅｒｉｓｉｍｐｏｒｔａｎｔｔｏｓｔｕｄｙｇｅｎｅｒｅｇｕｌａｔｉｏｎ，ｖｅｃｔｏｒｓｃｏｎｓｔｒｕｃｔｉｏｎａｎｄｔａｒｇｅｔｐｒｏｔｅｉｎｓｅｘｐｒｅｓｓｉｏｎ．Ｆｒｏｍｔｈｅｆｒｅｑｕｅｎｔｌｙｕｓｅｄｗａｙｔｈａｔａｐｐｌｙｉｎｇｐｒｏｍｏｔｅｒｔｒａｐｐｉｎｇｆｏｒｃｈｏｏｓｉｎｇｐｒｏｍｏｔ－ｅｒｔｏｔｈｅｕｓｉｎｇｏｆＰＣＲ，ｔｈｅｒｅｗｅｒｅｌｏｔｓｏｆｗａｙｓｆｏｒｐｒｏｍｏｔｅｒｃｌｏｎｉｎｇ．Ａｆｔｅｒｗａｒｄｓ，ａｓｅｒｉｅｓｏｆｔｅｃｈｎｉｑｕｅｓｂａｓｅｄｏｎＰＣＲｆｏｒｐｒｏｍｏｔｅｒｃｌｏｎｉｎｇｓｕｃｈａｓＡ－ＰＣＲ，Ｉ－ＰＣＲ，ＬＡ－ＰＣＲ，ＴＡＩＬ－ＰＣＲｗｅｒｅｄｅｖｅｌｏｐｅｄｉｎｓｕｃｃｅｓｓｉｏｎ．Ｔｈｅｙｏｆ－ｆｅｒｅｄｍｏｒｅｒｅｌｉａｂｌｅａｎｄｒｅａｓｏｎａｂｌｅｗａｙｓｏｆｃｌｏｎｉｎｇｔｈｅｐｒｏｍｏｔｅｒ．Ｓｏｍｅｗａｙｓｏｆｃｌｏｎｉｎｇｐｌａｎｔｇｅｎｅｐｒｏｍｏｔｅｒｓｗｅｒｅｉｎｔｒｏｄｕｃｅｄ，ｍｅａｎｗｈｉｌｅｔｈｅｌｉｍｉｔａｔｉｏｎｏｆｔｈｅｓｅｗａｙｓｗａｓｉｎｔｒｏｄｕｃｅｄａｎｄｔｈｅｉｒｄｅｖｅｌｏｐｉｎｇｐｒｏｓｐｅｃｔｗａｓａｌｓｏｄｉｓ－ｃｕｓｓｅｄｉｎｔｈｉｓｐａｐｅｒ． ＫｅｙｗｏｒｄｓＰｒｏｍｏｔｅｒ，Ｐｒｏｍｏｔｅｒｔｒａｐｐｉｎｇ，Ｉ－ＰＣＲ，ＡｎｃｈｏｒｅｄＰＣＲ，ＴＡＩＬ－ＰＣＲ 启动子是ＲＮＡ聚合酶能够识别并与之结合、从而起始基因转录的一段ＤＮＡ序列，是基因表达调控的重要元件。启动子对外源基因的表达水平影响很大，是基因工程表达载体的重要元件。植物基因启动子的克隆，对研究植物基因表达调控和构建表达载体至关重要。本文就近几年来克隆植物基因启动子的各种方法做一综述，以期促进对启动子分离技术的应用。 １植物基因启动子克隆的几种方法 １．１利用常规ＰＣＲ技术克隆启动子 对于序列已知的启动子，只需要根据已知的启动子序列设计引物，然后采用ＰＣＲ扩增的方法就可以获得该启动子。该方法比较简便快捷，是近年来使用较为广泛的一种方法。 王利军等（２００４）根据ＧｅｎＢａｎｋ中拟南芥基因组序列中ａｔｓ１Ａ（核酮糖－１，５－二磷酸羧化酶小亚基）基因启动子的序列设计引物，以拟南芥总ＤＮＡ为模板，ＰＣＲ扩增出ａｔｓ１Ａ的基因启动子区片段，将其克隆到ｐＭＤ１８－Ｔ载体上，得到重组质粒并且进行测序，序列分析表明，所克隆的片段为１１１７ｂｐ，与Ｇｅｎ－Ｂａｎｋ中的ａｔｓ１Ａ基因启动子序列比较，发现只在－７７６位缺失１个碱基。 该方法简便、快捷、操作简单，但不能克隆到全新的启动子。

网络游戏公司简介范文3篇(完整版)

网络游戏公司简介范文3篇 网络游戏公司简介范文3篇 网络游戏指以互联网为传输媒介，以游戏运营商服务器和用户计算机为处理终端，以游戏客户端软件为信息交互窗口的旨在实现娱乐、休闲、交流和取得虚拟成就的具有可持续性的个体性多人在线游戏。下面是网络游戏公司简介范文，欢迎参阅。 网络游戏公司简介范文1 边锋网络游戏是201X年8月整合入盛大网络旗下的边锋游戏和201X年12月整合入盛大网络旗下的游戏茶苑两家中国领先的棋牌游戏公司合并运营而成的。201X年边锋公司购回了盛大持有的股份，独自进行边锋网络游戏的运营，运营的游戏平台有： 纸牌类，如： 德清点子、五人原子、四人斗地主、原子、六扣、双扣、三扣 一、跑得快、斗地主、德州扑克、升级、红五等; 棋类，如： 三英战吕布、军旗翻翻棋、爆笑四国、陆战棋、黑白棋、双飞棋、五子棋、飞行棋等; 骨牌类，如： 新沈阳麻将、丽水麻将、富阳麻将、合肥麻将、德阳麻将、攀枝花麻将、自贡麻将、杭州麻将等; 对战类，如： 台球、对对碰、宇宙方块、斯诺克、疯狂火箭、俄罗斯方块、挖哈哈、连连看等。

桌游类，如： 三国杀online等等 201X年4月，盛大又将边锋连同浩方以35亿元的高价出售给浙报传媒集团，其中，浙报传媒为边锋估值3 1.8亿人民币，而盛大当年收购边锋的总代价为201X万美元，约合 1.64亿元人民币，8年之间，边锋增值30多亿元。 据浙报传媒公告显示，201X年杭州边锋营业收入4亿元，净利润 1.44亿元;201X年营业收入 6亿元，净利润9946万元。 网络游戏公司简介范文2 上海盛大网络发展有限公司 盛大文学通过整合国内优秀的网络原创文学力量，推动纸质书出版，加强第三方版权内容的数字化运营，构建全球领先的正版数字书城，旨在推动数字出版，引领数字阅读潮流，为消费者提供包括数字图书、网络文学、数字报刊等数字商品。并依托原创故事，推动实体出版、影视、动漫、游戏等相关文化产业的发展。 盛大在线作为专为无物流的文化和虚拟产品提供数字出版的服务平台，致力于提供基于云计算服务的综合解决方案。通过完善的统一登录、计费、内容分发、广告营销、搜索、客户关系服务等，为广大互联网用户和企业获取数字内容产品提供优选渠道和专业化的用户服务体系。 盛大游戏是中国领先的网络游戏开发商、运营商和发行商，致力于打造中国乃至全球领先的网络游戏平台。盛大游戏拥有201X多名自

结核病工作职责-方案

督导管理人员的职责 1.应根据患者实际情况确定服药地点和时间，面视患者服药。 2. 患者如未按时服药，应及时采取补救措施。 3. 每次督导服药后按要求填写“肺结核患者治疗记录卡”。 4. 一旦发现患者出现不良反应或中断用药等情况，及时报告结防所。 5. 督促患者定期复查，协助收集痰标本。 6. 患者完成全程治疗后，应将“肺结核患者治疗记录卡”上交至乡镇卫生院，转送至县结防机构归档保存。

结核病工作实施方案 为了进一步加强本乡镇结核病防治工作，控制结核病的传播和流行，保障人民群众身体健康，促进经济和社会发展，结核本乡镇实际，制定本实施方案。 一、结核病流行与防治工作现状 结核病是严重危害人民身体健康的经呼吸道传播的慢性传染病，为全球所关注。我国是世界上22个结核病高负担国家之一，结核病患者数量居世界第二位结核病已被国家列为重点控制点的重大疾病之一。本乡镇结核病疫情的波动有几个特征：一是外来流动人口导致新发病例的增加、管理难度加大；二是家庭经济困难无法完成疗程；三是多耐药结核病的增加使结核病治愈变得更加复杂；四是结核病合并其他疾病的双重感染使结核病难以治愈。因此必须进一步加大结核病防治力度，全面有效的实施现代结核病控制策略，把结核病对人民健康的危害以及对本市经济和社会发展的影响降到最低限度。 指导原则 政府领导、部门合作、全社会参与、卫生部门管理，共同做好结核病防治工作。 坚持“预防为主，防治结合”的方针，加强结核病人发现和结核病人定点治疗 工作 因地制宜、条块结合，落实病人的督导管理和健康教育。 全面实施现代结核病控制策略，提高防治效果。 加强结核病防治宣传培训，提高市民健康知识知晓度和主动就诊意识。 （一）实行结核治疗费用“收、减、免”政策，控制传染源。 二、总体目标 （一）建立和完善本乡镇结核病防治工作的可持续发展机制。 （二）以乡镇为单位实行现代结核病控制策略的覆盖率达到１００％，并不断提高内涵质量。（三）完成本乡镇肺结核病人治疗任务。 三、工作指标 进一步减少结核病感染、患病和死亡，切实降低结核病疾病负担，提高人民群众讲课水平，促进国民经济发展和社会和谐稳定。 （一）村卫生室对可疑肺结核及肺结核病人的转诊率、活动性肺结核病人归口诊治率达到９5％以上；新发现活动性肺结核患者规则治疗率达到95％。 （二）新发现活动性肺结核病患者村负责督导管理的覆盖率达到８5％。 （三）新发现活动性肺结核患者的治愈率达到８５％以上。 四、主要措施 （一）健全结核病防治网络，提高结核病防治能力 根据预防归口、功能对应、防治结合、归口管理、各司其职的原则，不断加强本辖区疾病预防控制体系及结核病防治网络的建设，充实和稳定结核病防治专业技术队伍，提高防治技术水平。结核病诊治指定医疗机构必须按照“谁治疗、谁管理”的防治工作原则，落实结核病病人资料的规范管理，执行结核病标准化疗方案，加强对病人健康教育，实施定期配药、定期复查及服药监测等管理工作，确保治疗效果。 开展辖区内结核病人的化疗督导管理及健康教育。对辖区内新发现的活动性肺结核病人，必须明确专人承担化疗督导管理。病人所在的单位也要督促病人及时就诊，使病人得到全程、规范治疗。 （二）积极发现和治疗肺结核患者

优秀学生个人简历

优秀学生个人简历 姓名:XXX 性别：男 就读专业：人力资源 学号：XXX 宿舍号：6-626 籍贯：江苏省昆山市 出生年月：90.1 政治面貌：团员 原毕业学校：江苏省昆山市亭林中学 联系电话：xxx 在校期间任职情况： 时间班干部职务学生会职务 2005.9~2006.1（高一.上）劳动委员—— 2006.3~2006.6（高一.下）劳动委员劳动卫生部长2006.9~2007.1（高二.上）副班长兼劳动委员学生会主席2007.3~2007.6（高二.下）班长兼劳动委员学生会主席2007.9~2008.1（高三.上）副班长兼劳动委员学生会主席2008.3~2008.6（高三.下）劳动委员——

总结： 班干部方面：通过三年的班干部工作，给自己在管理能力等方面得到了很大的锻炼，也从中吸收了很多经验，平时工作时能积极主动配合班主任，勤沟通，认真贯彻、执行各项计划和决定，能发动、组织、带领全班同学开展各项活动，并且与学生相处友好。 学生会干部方面：能熟练地主持学生会日常工作及学生会的会议，对学生会的工作十分熟悉，能出色地完成学校下派的各项任务，曾多次被评为校级、昆山市级“优秀学生干部”称号，也夺得了学校领导老师及广大学生的一致好评。 在校期间所获荣誉： 时间奖项 2006年度第一学期在“班十佳”评选中获“班级工作好”称号； 2006年度第二学期获校“优秀学生干部”称号； 2006年度第二学期在优秀学生会干部评选中获“优秀学生会干部”； 2007年度第一学期获校“三好学生”； 2007年度第二学期在“班十佳”评选中获“班级工作好”称号； 2007年度第二学期获校“三好学生”； 2007年度第二学期在优秀学生会干部评选中获“优秀学生会干部”； 2007年6月被评为昆山市“优秀学生干部”； 2008年度第一学期获校“优秀学生干部”称号； 2008年3月在昆山市青少年法制宣传教育活动中，被评为“百佳学法小标兵”；

结核病防治知识培训内容

结核病防治知识培训内容 一、结核病疫情现状 结核病是经呼吸道传播的慢性传染病，主要发生肺部。结核病在全球广泛流行，严重危害了人民群众的身体健康，以成为重大的公共卫生问题和社会问题。我国是世界上22个结核病高负担国家之一，结核病患者数量居世界第二。我县目前有活动性肺结核患者约1500人，其中传染性肺结核患者约500余人。严重制约了我国经济和社会发展。 二、结核病的相关政策 免费检查的范围：肺透视、X 胸片和痰涂片检查(2次拍片、4次查痰）。 免费治疗范围包括：6——8个月统一方案的抗结核药和护肝药。特殊病人可享受2次（14个月药）。其它费用仍需自付。 三、结核病的基本知识 结核病是经呼吸道传播的慢性传染性疾病，主要通过病人咳嗽、打喷嚏好、大声说话时喷出的飞沫传染给他人。一般来说被感染并不一定发病，当身体抵抗力下降时，才能进展为结核病。连续咳嗽、咳痰2周以上，或痰中带血丝应怀疑得了结核病。其它症状还有低烧、夜间盗汗、疲乏无力、体重减轻等。正规治疗90%的结核病可以治愈。 四、发现病人

首诊医生要题高对结核病可疑症状者的警觉性，发现可疑结核病要填写《初诊病人登记本》，记录的病人资料要真实可靠，以备核查。及时报告。网络直报人员认真核对疫情信息，确保疫情信息准确性和完整性。 五、转诊 接诊医生在填写传染病报告卡的同时，填写结核病人转诊单。一式三份：一份由公共卫生科备案，一份由公共卫生科送达县结控项目办，一份由病人携带，到指定结核病专家门诊就诊。 六、追踪病人 各村接到上级的通知，可用电话追踪、直接追踪。两种方法。要在三天之内将病人追踪到位。 七、病人的系统管理 1、查痰时间：初治涂阳病人（含种症涂阴病人）在疗程满 2.5.6. 个月时，复治涂阳病人在疗程满2.5.8. 月时，各查痰一次。初治、复治涂阳病人在疗程满2个月痰仍为阳性者，应在治疗满3个月时增加查痰一次。 2、查痰要求：初治病人应送3份标本（即时痰、夜间痰和清晨 痰）。随访病人每次两份标本（即时痰、夜间痰）。痰量3——5毫升。

植物逆境相关启动子及功能

HEREDITAS (Beijing) 2010年3月, 32(3): 229―234 ISSN 0253-9772 https://www.sodocs.net/doc/185449689.html, 综 述 收稿日期: 2009?09?13; 修回日期: 2009?10?28 基金项目:国家自然科学基金项目 (编号：30871389)和辽宁省优秀人才项目(编号：2009R36)资助 作者简介:朱丽萍(1984?), 女, 硕士, 专业方向：植物分子生物学及基因工程。E-mail: zhuliping0612@https://www.sodocs.net/doc/185449689.html, 通讯作者:李秋莉(1969?), 女, 博士, 教授, 研究方向：植物分子生物学及基因工程。E-mail: liqiuli@https://www.sodocs.net/doc/185449689.html, DOI: 10.3724/SP.J.1005.2010.00229 植物逆境相关启动子及功能 朱丽萍, 于壮, 邹翠霞, 李秋莉 辽宁师范大学生命科学学院, 大连 116029 摘要: 启动子是调控基因表达的重要顺式元件, 在植物基因表达调控过程中起着重要作用。目前植物抗逆基因 工程中, 人们大多使用组成型表达启动子驱动目的基因的表达。组成型表达启动子虽然能提高转基因植株的抗逆性, 但是其持续过量地表达转化的外源基因会阻碍植物的生长且减少其产量。因此, 只在胁迫条件下才会驱动外源基因表达的诱导型启动子的研究显得尤其重要, 已成为目前研究的热点。文章综述了受非生物逆境和生物逆境胁迫诱导的植物基因启动子的种类和功能, 并展望了植物逆境诱导启动子的研究方向和前景。 关键词: 逆境; 诱导型启动子; 功能分析 Plant stress-inducible promoters and their function ZHU Li-Ping, YU Zhuang, ZOU Cui-Xia, LI Qiu-Li College of Life Sciences , Liaoning Normal University , Dalian 116029, China Abstract: Promoter is an important cis -regulatory element for gene expression and plays an important role in the process of plant gene expression and regulation. Constitutive promoters are being used to drive alien gene expression in most trans-genic engineering. Although constitutive promoters can improve resistance of transgenic plants to abiotic stresses, over- and constitutive-expression of the alien genes have been shown to cause stunted growth and reduction of yield in transgenic plants. Therefore, inducible promoters, which are expressed only when exposed to stresses, are of importance. This paper reviews the types and functions of plant gene promoters induced by bio- and abio-stresses. The prospect of stress-induced promoters was discussed. Keywords: adversity; stress-inducible promoter; function analysis 植物逆境是指对植物施加有害影响的环境因子, 对植物产生重要影响的逆境主要有缺水、低温、盐碱、高温等非生物逆境, 以及病原等生物逆境。无论非生物逆境还是生物逆境都会造成各种农作物产量和质量的下降, 因此, 培育和推广抗逆品种是保证作物稳产高产的有效途径。利用植物自身的抗逆基因, 通过常规育种和分子标记辅助育种方法可以培育抗逆新品种, 然而, 抗性基因在种属间的利用 具有一定的局限性。转基因方法可以克服上述局限, 为作物抗逆育种开辟一条新途径。 植物基因调控主要是在转录水平上进行的, 受多种顺式作用元件和反式作用因子的相互协调。植物基因启动子是重要的顺式作用元件, 它是位于结构基因5′端上游区域调控基因转录的一段DNA 序列, 能活化RNA 聚合酶, 使之与模板DNA 准确地结合, 确保转录精确而有效地起始, 是转录调控的

快递公司简介范文

快递公司简介范文 中国快递行业目前处于国内快递行业和国际快递巨头竞争激烈的环境中，相对国际快递巨头，中国民营快递公司处于比较弱势，中国国内快递企业多争夺于底端市场。中国快递业务发展程度还很低，现在得快递业务量还不到GDP的0.3%，与发达国家达到GDP的1%左右相比差距很大。下面是快递公司简介范文，欢迎参阅。 快递公司简介范文1 80后快递服务有限公司，是以服务为主体的公司。服务的范围包括有同城快递，物流配送，年节送礼，同行调货，门市宅长期配送服务。另外我们还计划推出80后商务套餐。以满足江城商务迅猛发展的快捷生活需求。 公司名称：武汉80后快递服务有限公司所属行业：快递，服务业企业性质：集体企业成立日期：20xx-4-30武汉80后快递服务有限公司公司的服务网络计划在两个月内完成建设，下一步招募专业人员组建一个为80后为主要人群服务的心理援助中心，帮助解决80后为主要人群在工作，学习，生活，恋爱，婚姻及家庭子女教育中遇到的各种问题。 快递公司简介范文2 申通快递 公司注册商标为“STO+申通”，注册编号为1379930。主要承接非信函、样品、大小物件的速递业务。20xx年3月公司通过ISO9001:20xx国际质量管理体系认证。 公司奉行“团结、务实、开拓、创新”的企业精神，“快速、准确、安全、周到、”的服务方针公司经营十余年来，已深得广大客户的信任和支持。 公司自1993年成立以来，在董事长、总经理陈德军的正确领导下，在广大客户的支持和关怀下，在全体员工的艰苦奋斗和顽强拼搏下，先后荣获上海市松江区民营企业20xx至20xx年度的《信得过企业》、《先进企业》荣誉称号;20xx年，公司荣获《中国物流十大影响力品牌》称号，公司董事长、总经理陈德军先生个人荣获《中国品牌建设十大杰出企业家》荣誉称号。 申通快递介入电子商务配送业务已经开始起步，并计划为新业务斥资千万，一套全新的标准化流程和服务标准已经设计完毕，软件系统也已具备代收货款功能，与几大电子商务网站的谈判正在进行。

结核病防治实施措施

结核病防治实施措施 发表时间：2014-04-02T16:53:56.297Z 来源：《医药前沿》2014年第1期供稿作者：骆天娥白忠婷[导读] 结核病是严重危害人类健康的慢性传染病，1998年WHO再次指出“遏制结核病行动刻不容缓”我国是全球结核病高发国之一 骆天娥白忠婷 （新疆兵团第五师九0团医院新疆博乐 833409）【摘要】目的：提高结核病防治实施质量，实现联合国千年发展目标，降低结核病疫情，提高全民健康水平，确保社会经济发展。方法：健康促进人员培训痰涂片检查与质量控制，合理用药治疗。结果：自2003年至2013年我院开展结防工作扎实有效，发病率递减，无复发率无死亡率。结论：认真落实结核病防治措施，可以达到降低患病率，控制传染源，减少发病。 【关键词】结核病实施措施 【中图分类号】R52 【文献标识码】A 【文章编号】2095-1752（2014）01-0090-02 结核病是严重危害人类健康的慢性传染病，1998年WHO再次指出“遏制结核病行动刻不容缓”我国是全球结核病高发国之一，疫情如下：①高患病率②高耐药率③高死亡率④高感染率⑤低递降率⑥农村疫情高于城市⑦青壮年结核病患病和死亡比例高，因此做好结核病防治工作势在必行。 1、健康促进：结核病防治健康促进是指通过对结核病防治政策与结核病防治知识的宣传与倡导，动员全社会相关部门、相关力量和相关资源，解决有关结核病防治存在问题的一种社会行动。 1.1目标：提高不同目标人群对结核病防治政策和防治知识的认识，改变其陈旧，错误的观念和认识，使之采取正确的行为或改变不正确的行为，提高各级领导对结核病防治工作重要性的认识，加大对结核病防治工作的投入，使得有结核病可疑症状的人能够及时就诊，结核病患者能够配合医生的督导治疗。 1.2步骤：开展任何一项健康促进活动，必须长期有效科学实施，包括明确活动目标，制定活动基本框架，制作宣传材料，组织实施监控与评价，并做好各项活动原始资料的记录和管理。1.3提供结防核心信息：①肺结核是一种严重危害人类健康的慢性呼吸道传染病;②咳嗽咳痰2周以上或痰中带血丝应怀疑得了肺结核;③怀疑得了肺结核应到居住地结防机构接受检查和治疗;④在结防机构检查和治疗肺结核可享受国家免费治疗;⑤坚持正规治疗，绝大多数肺结核患者可以治愈。 2、人员培训：结核病防治培训工作，是提高结防机构人员素质，改善执行能力，确保结核病防治工作质量的重要措施，通过多种方式对各级结防机构业务人员和医疗单位有关人员进行管理和技术方面的培训，提高和改善结核病防治人员的管理水平和业务技术水平，不断提高实施能力和对结核病患者的服务水平，培训应遵循统筹协调，逐级培训，理论与实践相结合，培训方式多样化的基本原则。 2.1培训对象：①各级结防机构专业人员包括负责规划管理，患者发现及治疗管理，统计监测药品管理，痰菌检验，督导等工作人员。 ②医疗卫生机构包括疾控中心，内科、放射科、检验科及乡镇医生工作人员。 2.2培训内容：①结防机构人员：结核病的诊断，鉴别诊断，治疗与管理，统计监测，药品管理，督导和实验室检查与质控等。②医疗卫生机构医务人员：结核病防治规划管理，结核病的诊断与鉴别诊断，结核病疫情报告与转诊。③乡镇医生：结核病患者发现及治疗管理，健康教育，疫情报告等。 3、痰涂片检查与质量控制：从事基层检验室工作26年，深知痰涂片检查是肺结核病人诊断与鉴别诊断，制定治疗方案、疗效考核的主要手段，做好痰涂片检查，加强其质量控制，是做好结核病控制工作的重要保证。 3.1对象：①疑似肺结核病人，结核病可疑症状者，（如咳嗽咳痰2周以上，或痰中带血）;②接受结核治疗的病人，治疗期间按规定定期做痰菌复查。 3.2要求：①初诊病人应送3份痰标本（夜间痰、清晨痰、即时痰）复诊随访病人按期送检2份痰（清晨痰、夜间痰）②痰标本的采集：应以脓样、干酪样或粘液痰为合格标本，痰量3-5毫升，由检验人员验收痰标本，痰液不合格者应要求重新送检。③痰标本的保存：痰标本容器应密封，防止痰液外溢，不能立即做涂片检查的标本，须放置4度冰箱保存，防止痰液干涸或污染。 3.3痰涂片质量控制：①室内质控：包括痰标本收集、染色剂质量、涂片制备、染色技术、显微镜维护及镜检，结果登记以及痰片的保存均应有质控记录备查。②室间质量控制：包括盲法复查，实验室现场评估，盲法评价是评价整个实验室操作水平，包括检查标本的质量，涂片的大小厚薄，染色质量等，以便采取纠正措施，提高实验室工作质量，实验室现场评估由上级经验丰富的技术人员担任，对下级实验室每年进行2次现场评估指导。 4、合理用药治疗：肺结核患者确诊后要及时给予治疗，合理用药是消除传染性，阻断传播和治愈患者的关键措施。 4.1免费治疗对象：初治活动性肺结核患者及复治涂阳结核患者，为其提供免费抗结核治疗药品。 4.2治疗方式：对肺结核患者以不住院治疗为主，对少数危急、重症患者、伴有严重合并症或并发症的肺结核患者可采取住院治疗，出院后转至结防机构继续实施严格治疗管理，直到疗程结束，治疗期间严格管理控制药品种类、计量、用法和不良反应。通过实施以上系列措施，我院结防工作初见成效，社区居民对结核病的危害性、预防、检查及免费治疗等知晓率大幅度提升，主动咨询检测人员逐年递增，痰涂阴患者完成全部疗程，痰涂阳患者治愈率85%以上。但结防工作任重而道远，需不断完善可发展机制，努力提高现代化结核病控制实施质量，认真落实，常抓不懈。确保人民健康，社会经济繁荣发展。参考文献 [1]中国结核病防治规划实施工作指南（2008年版）.

网络科技公司简介范文5篇

网络科技公司简介范文5篇Introduction of network technology company 编订：JinTai College

网络科技公司简介范文5篇 小泰温馨提示：写作是运用语言文字符号以记述的方式反映事物、表达思想感情、传递知识信息、实现交流沟通的创造性脑力劳动过程。本文档根据写作活动要求展开说明，具有实践指导意义，便于学习和使用，本文下载后内容可随意修改调整修改及打印。 本文简要目录如下：【下载该文档后使用Word打开，按住键盘Ctrl键且鼠标单击目录内容即可跳转到对应篇章】 1、篇章1：网络科技公司简介范文 2、篇章2：网络科技公司简介范文 3、篇章3：网络科技公司简介范文 4、篇章4：网络科技公司简介范文 5、篇章5：网络科技公司简介范文 网络公司不仅仅是提供域名注册、空间租用、网站开发、网站建设与网络营销活动策划相关的企业组织。下面是网络科技公司简介范文，欢迎参阅。 篇章1:网络科技公司简介范文

支付宝（xxx有限公司是国内领先的独立第三方支付平台，是阿里巴巴集团的关联公司。支付宝致力于为中国电子商务提供“简单、安全、快速”的在线支付解决方案。 支付宝公司从20xx年建立开始，始终以“信任”作为产 品和服务的核心。不仅从产品上确保用户在线支付的安全，同时让用户通过支付宝在网络间建立起相互的信任，为建立纯净的互联网环境迈出了非常有意义的一步。 支付宝提出的建立信任，化繁为简，以技术的创新带动 信用体系完善的理念，深得人心。在六年不到的时间内，为电子商务各个领域的用户创造了丰富的价值，成长为全球最领先的第三方支付公司之一。截止到20xx年12月，支付宝注册用户突破5.5亿，日交易额超过25亿元人民币，日交易笔数达 到850万笔。 支付宝创新的产品技术、独特的理念及庞大的用户群吸 引越来越多的互联网商家主动选择支付宝作为其在线支付体系。 目前除淘宝和阿里巴巴外，支持使用支付宝交易服务的 商家已经超过46万家;涵盖了虚拟游戏、数码通讯、商业服务、机票等行业。这些商家在享受支付宝服务的同时，还是拥有了一个极具潜力的消费市场。

学校结核病防治应急处理预案

学校结核病防治应急预案 为进一步做好我校的结核病防治工作,确保全体师生身体健康和生命安全，规范学校结核病散发病例管理措施，妥善处置学校群体性结核病感染事件，努力维护校园和社会稳定。根据《传染病防治法》和《突发公共卫生事件的应急条例》的各项要求，我校将积极做好学校结核病防治工作，提高学校防控结核病的应急能力，制订本预案。 一、严格组织领导 学校成立防控传染病工作领导小组，全面负责学校的各项防控工作。 组长：侯桂军 对学校结核病防治工作负总责，具体落实人员分工、资金保证、部门协调。 副组长：罗艳 协助组长做好相关事务工作。 组员： 许铁利 具体负责学校卫生监督，防控必须物品的采购，校园消毒。 周华 具体负责结核病防治知识宣传工作及资料汇总。 班主任

具体负责本班的卫生打扫，结核病防治知识教育、学生晨检及缺勤统计、学生突发事件处理及报告。 二、学校结核病预防措施 1、加强健康教育，通过课堂教学、专题讲座、黑板报、宣传栏等形式，广泛进行宣传教育，提高学生认知水平，增进其自我防护意识和能力。 2、教室、图书室等人群聚集场所要特别注意卫生和通风，保持空气流通。 3、教育学生养成良好个人卫生习惯，不随地吐痰，咳嗽、打喷嚏时掩住口鼻，常洗澡、勤换衣被等。 4、广泛开展阳光体育活动，加强体育锻炼，增强学生体质。 5、定期进行健康体检，建立学生健康档案，对有结核病史及有结核病家族史，体质较差学生，要注意重点观察，发现异常及时诊治。 三、学校发生肺结核病的处置办法 （一）对疫情的处置 1、学校一旦发现首例肺结核病患者或疑似病例，应在24小时内将疫情上报疾控部门和教育主管部门，不得延报、瞒报。 2、积极协助疾控部门开展流行病学调查，查清疫情的来龙去脉。如果传染源在学校内，应迅速采取措施消除传染源。 3、当疫情呈个别散发状态，经疾控部门认定无须停课时，应注意一方面做好患病学生的隔离、治疗工作，另一方面对密切接触者进

植物组织特异性启动子的研究进展

植物组织特异性启动子的研究进展 摘要：组织特异性启动子也称器官特异性启动子(organ specific promoter)，可调控基因只在某些特定的部位或器官中表达，并往往表现出发育调节的特性。本文介绍了植物组织特异性启动子中的花、果实、种子及叶特异性启动子，并对植物组织特异性启动子研究中问题进行了讨论和展望。 关键词：植物组织特异性启动子，研究进展 启动子(promoter)是RNA聚合酶能够识别并与之结合从而起始基因转录的DNA序列，是重要的顺式作用元件，位于结构基因5’端上游区的DNA序列，能指导全酶与模版的正确结合，活化RNA聚合酶，并使之具有起始特异性转录的形式，决定转录的方向和效率以及所使用的RNA聚合酶类型，是转录调控的中心。目前，植物基因工程中常用的启动子为组成型表达启动子(Constitutive promoter)。组织特异性启动子也称器官特异性启动子(organ specific promoter)，可调控基因只在某些特定的部位或器官中表达，并往往表现出发育调节的特性。外源基因在细胞中表达是基因工程研究的关键，而组织特异性启动子不仅能使目的基因的表达产物在一定器官或组织部位积累，增加区域表达量，同时也可以避免植物营养的不必要浪费。因此，组织特异性启动子一直都是植物基因工程中研究的重点和难点，研究者在植物的分生组织、维管束组织、薄壁组织、花粉、种子和胚乳等几乎各种组织中都发现有组织特异性驱动基因表达的启动子[1]，尤其在根、维管束和韧皮部特异表达启动子研究中取得了很大进展。 1 植物生殖器官表达的组织特异性启动子 1.1 花特异启动子 高等植物发育过程中花器官的形成是一个十分复杂的过程，它包括一系列器官分化及严格控制的细胞及生化变化，同时伴随大量基因的协同表达。植物花特异表达启动子只在植物开花的时期启动基因的表达，它的分离克隆为花卉的品质改良提供了重要的顺式调控元件。 人们克隆了许多花药特异表达的基因，如在花药绒毡层特异表达的TA29[2]和A9[3]以及在花粉壁特异表达的Bp4A[4]基因等，这些基因都是在花药营养细胞中表达，与生殖细胞的分化无关。Mariani等将烟草花药绒毡层特异表达基因启动子TA29分别与核酸酶Bamase和RnaseTl基因融合后转化植物，这两种核酸酶基因在花药中特异表达，破坏绒毡层，获得雄性不育烟草和油菜[5,6]，开创了基因工程创造雄性不育系的先河，至今已在烟草[7]、油菜[8]等植物上获得成功。还有在花药中特异表达的基因，如拟南芥和油菜的APG 基因等。在花粉中特异表达的基因如：玉米的Zm13 (蛋白酶抑制剂)、CDPK （钙依赖型蛋白激酶）、番茄的LAT52、LAT 59及油菜Bp4 、I3 、E2和F2s等。 近年来人们相继分离和鉴定了许多花特异表达基因的启动子，如矮牵牛和金

我国结核病疫情现状

．我国结核病疫情现状 结核病是由结核杆菌感染引起的慢性传染病。结核菌可能侵入人体全身各种器官，但主要侵犯肺脏，称为肺结核病。结核病是一种古老的传染病，历史上对人类的危害触目惊心。解放前，民间称结核病为痨病，并且有“十痨九死”的说法。 　自20世纪90年代以来结核病在全球“死灰复燃”，许多国家包括结核病疫情控制较好的国家，不同程度地出现了疫情下降缓慢或严重反弹的局面，发病率以每年1.1％的速度增长，结核病再次成为威胁人类健康的主要传染病，成为严重的公共卫生问题和重大的经济社会问题。据世界卫生组织估计，全球每年新发结核病患者880万例，其中传染性结核病患者390万例，每年因结核病死亡的患者约200万人。面对全球结核病疫情日趋严重的局面，世界卫生组织将结核病列为重点控制的三种传染病之一，并于1993年宣布全球结核病处于紧急状态，强调遏制结核病的流行已经到了刻不容缓的程度。 　我国是世界上22个结核病高负担国家之一，患者人数仅次于印度居全球第二位。世界卫生组织2004年召开的第二届全球遏制结核病伙伴论坛大会上，将我国列在需要特别引起警示的国家和地区的首位，具体表现为6“多”：一是感染人数多，全国有多达5.5亿的人口感染过结核菌，约占全国人口的45％，明显高出全球平均感染水平；二是患病人数多，全国活动性肺结核、传染性肺结核患病率分别为367／10万和122／10万，有活动性肺结核患者约450万，其中传染性肺结核患者约150万；三是新发患者多，全国每年新发生活动性肺结核患者约145万，其中传染性肺结核病人65万例；四是死亡人数多，全国每年约有13万人死于结核病；五是农村患者多，全国约80％的结核病患者集中在农村，而且主要在经济不发达的中西部地区；六是耐药患者多，全国结核病耐药率高达28％。 　我国部分地区结核病疫情回升的原因是多方面的，有流动人口骤增、耐药结核菌的蔓延、结核菌与艾滋病病毒的双重感染等客观原因，更主要的是，一些地方政府对结核病疫情的严重性和结核病控制工作的重要性认识不足，缺乏落实政府承诺的自觉性；对结核病防治经费的投入严重不足，忽视了结核病防治机构及能力的建设；相当一些地方未能把有效控制结核病流行纳入经济社会发展规划之中，相关部门职责不明、参与不够；未能全面、准确、有效地推行与落实现代结核病控制策略，患者发现率低，督导化疗留于形式；普遍对结核病健康教育重视不够，结核病防治的相关知识不普及等。 二．传播途径

系统集成公司简介范文

系统集成公司简介范文 系统集成商是指具备系统资质，能对行业用户实施系统集成的企业。下面是系统集成公司简介范文，欢迎参阅。 系统集成公司简介范文1 广州系统集成公司，专业为客户提供结构化布线系统、网络技术工程、程控交换机系统安装、监控安防系统、一卡通系统、音视频系统、机房建设等系统方案设计、施工及维护的服务。 “全面满足，不断超越，永创新高，打造行业领跑者形象”，公司一直秉承“以市场为导向、以客户为中心”的发展理念，以“团结、务实、拼搏、创新”为宗旨，不断苦练内功，随时为广大客户提供最优质的产品与服务。 系统集成公司长久以来一直努力的目标，就是协助客户建立最具竞争力的信息化系统，即协助客户去规划、建设和维护高性能的网络系统、可靠的网络安全建设、智能建筑系统等。并在业界树立了良好的口碑和有了很好的发展。如今，开建智能的服务网络覆盖多个地方并都设有办事机构。自建立以来，开建智能坚持的目标从不曾改变，凭借着其日益成熟的经营理念和专业水平，开建智能必将协助客户获取更强的竞争力。 专业而经验丰富的技术人力资源。开建智能的全体员工拥有专业的技术知识，并在大型系统、结构化网络系统、远程通讯、办公自动化、系统技术支持，和软件编写方面拥有丰富的经验。

系统集成公司简介范文2 中国电信集团系统集成有限公司成立于1996年，是中国电信集团公司的全资子公司。公司旨在为大客户提供ICT整体解决方案、为电信运营商提供应用软件开发和IT服务支撑、为中小企业客户提供综合信息化服务。 公司依托于中国电信全国垂直一体化的三级营销服务体系和运行维护体系，凭借中国电信丰富的网络资源、专业的电信及IT技术、优秀的技术团队、广泛的客户资源和行业知识，致力于为电信运营商、政府、金融、企业提供网络基础设施建设、网络升级及改造、网络管理服务、网络及设备代维服务、设备租赁、应用软件集成及开发、IT 服务支撑等“一站式”服务。 公司在为电信运营商、全国性大客户进行一系列大型网络建设和服务的过程中，归纳总结了一整套项目管理方法，形成了独特、完善的项目管理体系和实力强大的核心团队。公司通过了ISO9001(2000)质量管理体系认证。同时，还获得了信息产业部颁发的“计算机信息系统集成一级资质”和“通信信息网络系统集成甲级资质”，是国内第一家拥有“双一级”资质的系统集成企业。 公司将站在客户的角度思考客户的业务运营，通过对客户业务运营流程以及信息化需求的全面理解，为客户提供创新而适用的综合信息化解决方案和ICT支撑服务，提升客户价值，与客户共同成长。 系统集成公司简介范文3 联通系统集成有限公司是中国联通的全资子公司，注册资金亿元，

爱心幼儿园结核病防控工作措施

2013年秋爱心幼儿园关于进一步 加强学校结核病防控预案 一、爱心幼儿园结核病防控领导小组 组长：张锦玉 副组长：刘燕 组员：冯静江奇枫徐敏江敏钱丽丽 李文国施金霞 二、具体分工 组长：张锦玉（总体负责） 副组长：刘燕（具体分管） 后勤主任：冯静（督查值周领导如何具体开展幼儿园周防控结核病工作。） 教学主任：江奇枫（督查幼儿园具体各部门防控工作落实情况，信息上报。） 教研组长：徐敏（督查防控结核病教育与宣传工作。）教研组长：江敏（对班主任、保育教师及时培训与指导。） 安保主任：钱丽丽（联系协调卫生部门，具体对各班工作落实指导并督查。） 班主任：（落实晨检和晚检，及时给安全办公室上报信息，具体做好结核病健康教育，及时科学处置生病幼儿。）门卫：李文国（把好师生、外来人员进出校门关，严格执行门卫工作制度。） 食堂责任人：施金霞（落实食品安全措施，让师生安全放心就餐。） 三、具体措施 （一）结核病常规预防学校措施 1.开展结核病健康教育。通过健康教育课、主题班会、宣传展板、黑板报、宣传窗，或开展讲座、播放影像制品等形式，对全体幼儿家长和教职员工广泛宣传结核病防治的核心知识，提高结核病的认知水平，增强自我防护意识，减少对结核病患者的歧视。核心知识包括： （1）肺结核是一种慢性呼吸道传染病； （2）咳嗽、咳痰2周以上，或痰中带血丝，应当怀疑得了

结核病； （3）得了结核病，应当到结防机构接受检查和治疗；（4）在结防机构检查和治疗肺结核，可享受国家免费政策； （5）只要坚持正规治疗，绝大多数肺结核患者是可以治愈的; （6）咳嗽、打喷嚏掩口鼻; （7）不随地吐痰; （8）出现肺结核可疑症状或被诊断为肺结核后，应当主动向学校报告，不隐瞒病情、不带病上课; （9）养成开窗通风习惯； （10）保证充足的睡眠，合理膳食，加强体育锻炼，提高抵御疾病的能力。 2.创建良好的幼儿园卫生环境。做好校园环境的清扫保洁，消除卫生死角。特别要做好教室、午睡室、图书馆(阅览室)、食堂等人群聚集场所的保洁和通风，保持室内空气流通。 3.落实幼儿园健康体检、晨检及因病缺课登记和追踪制度。 （1）按有关规定将结核病的检查项目作为幼儿新生入学体检和教职员工每年常规体检的必查项目，并纳入幼儿和教职员工的健康体检档案。对于通过幼儿园健康体检发现的疑似结核病病例，幼儿园应当及时告知家长到结核病防治机构检查确诊。 （2）落实由班主任或班级卫生员负责的晨检工作，重点了解每名幼儿是否具有咳嗽、咳痰、发热、盗汗等肺结核可疑症状。发现肺结核可疑症状者后，应当及时报告学校医务室，告知家长及时到结核病防治机构检查确诊。 （3）落实因病缺课登记和追踪制度。班主任应当及时了解幼儿的缺课原因。如怀疑为肺结核，应当及时报告幼儿园保健室，并由幼儿园保健室追踪了解学生的诊断和治疗情况。 （4）加强疫情报告。对幼儿园通过健康体检、晨检等途径发现的结核病疑似病例，疫情报告人应当及时向所在地医疗卫生机构和疾病预防控制中心报告。 （5）建立健全园内有关部门之间、幼儿园与家长之间、幼儿园与当地医疗卫生机构及教育行政部门之间的联系机制，明确具体联系人和联系方式。 （二）、幼儿园结核病散发病例管理措施 幼儿园结核病散发病例是指在学校内发现结核病确诊病例，但未达到结核病突发公共卫生事件级别。应当在强化