3.2 Enhancement of phytoremediation of Cd- and Pb-contaminated soils
Enhancement of phytoremediation of Cd-and Pb-contaminated soils by self-fusion of protoplasts from endophytic fungus Mucor sp.
CBRF59
Zujun Deng a ,?,Renduo Zhang b ,Yang Shi b ,Li’ao Hu b ,Hongming Tan c ,Lixiang Cao c ,?
a
School of Basic Courses,Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances,Guangdong Pharmaceutical University,Guangzhou 510006,PR China
b
School of Environmental Science and Engineering,Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology,Sun Yat-sen University,Guangzhou 510275,PR China c
School of Life Sciences,Sun Yat-sen University,Guangzhou 510275,PR China
h i g h l i g h t s
"The fusant T3was constructed by
self-fusion of protoplasts from Mucor sp.CBRF59.
"
The highest Cd concentration
tolerated by T3is 4-folds higher than that of CBRF59.
"
The fusant T3improved the
g r a p h i c a l a b s t r a c t
Effect of CBRF59and the self-fusant CBRF59T3on the extraction amount of Cd in rape shoots.Different small letters on the bars indicate that the multiple comparison differences are signi?cant with different fungi treatments and the same soils (P <0.05).The vertical line on each bar shows the standard deviation (n =3).CK represents the control soil without additional metals;Cd25,Cd50,Cd25+Pb500,and Cd50+Pb1000represent soil samples supplemented with 25,50mg kg à1of Cd(II),25mg kg à1of Cd(II)and 500mg kg à1of Pb(II),50mg kg à1of Cd(II)and 1000mg kg à1of Pb(II),
respectively.
a r t i c l e i n f o Article history:
Received 16September 2012
Received in revised form 27November 2012Accepted 28November 2012
Available online 25December 2012Keywords:
Endophytic fungus Mucor sp.
Self-fusion of protoplast Phytoremediation
a b s t r a c t
The aim of this study was to isolate protoplasts from endophytic fungi and to carry out self-fusion of protop-lasts for their enhancement of metal resistance.Self-fusant CBRF59T3with resistance to 25mM Cd(II)was con-structed by self-fusion of inactivated protoplasts from Mucor sp.CBRF59.The inoculation of CBRF59and
CBRF59T3improved signi?cantly the availability of Cd(II)and Pb(II)in the https://www.sodocs.net/doc/091951219.html,pared with CBRF59,CBRF59T3inoculation increased the content of water-soluble Cd(II)by 24%.The dry weight of rape inoculated with CBRF59and CBRF59T3was both higher than that of the uninoculation rape.Inoculation of CBRF59T3fur-ther increased the dry weight of rape by 62%than CBRF59in the higher Cd(II)-+Pb(II)-contaminated https://www.sodocs.net/doc/091951219.html,-pared with CBRF59,CBRF59T3inoculation increased the concentration of Cd(II)in rape shoots by 35–189%in Cd(II)-and Cd(II)-+Pb(II)-contaminated soils.The inoculation of CBRF59T3also enhanced the translocation of Cd(II)from roots to shoots and increased the amount of extracted Cd(II)by rape.The results indicated that the mutants constructed by protoplast fusion is a feasible and ef?cient method to improve stress tolerance of uncharacterized fungi for phytoremediation of soils contaminated by heavy metals.
ó2012Elsevier Ltd.All rights reserved.
0045-6535/$-see front matter ó2012Elsevier Ltd.All rights reserved.https://www.sodocs.net/doc/091951219.html,/10.1016/j.chemosphere.2012.11.065
Corresponding authors.Tel.:+862039352196;fax:+862039352186(Z.Deng),tel.:+862084110238;fax:+862084036215(L.Cao).
E-mail addresses:dengzujun66@https://www.sodocs.net/doc/091951219.html, (Z.Deng),caolx@https://www.sodocs.net/doc/091951219.html, (L.Cao).
1.Introduction
Among the methods to remediate heavy metal contaminated soils,phytoremediation is recognized as a cost-effective method compared with the conventional technologies,such as soil replace-ment,solidi?cation,and washing strategies.However,there are some critical problems in phytoremediation,such as phytotoxicity and limited contaminant https://www.sodocs.net/doc/091951219.html,bining increased plant avail-ability and reduced internal bioavailability of metals should allow plants to accumulate higher amount of metals without increasing phytotoxicity(Weyens et al.,2010).To reduce internal metal bio-availability and metal phytotoxicity in consequence,bacteria, which are equipped with a metal resistance or sequestration sys-tem leading to bioprecipitation of metals on the bacterial cell wall, can be inoculated into plants(Kotrba et al.,1999;Valls and de Lorenzo,2002;Wu et al.,2006).
Bioaugmentation with endophytic bacteria should have the advantage of the traditional bioaugmentation.Endophytic bacteria are less susceptible to predation and the host plant provides nutri-ents to the bacteria,supporting their growth and establishment (Weyens et al.,2009).Engineered endophytic bacteria have been used to improve phytoremediation of contaminated soils by organ-ic pollutants and/or toxic metals(Barac et al.,2004;Weyens et al., 2009,2010).
Compared with bacteria,fungi have not been fully exploited for bioremediation despite being dominant in the living biomass in soils and abundant in aqueous systems(Harms et al.,2011).Fungi have been de?ned as eukaryotic,heterotrophic,absorptive organ-isms,which typically develop a branched,tubular body called a mycelium and reproduce by means of sporulation.Filamentous fungi have evolved a spatially extensive growth form of hyphae, and the soil facilitates the development of?lamentous fungi be-cause of little mechanical disturbance in the medium(Harms et al.,2011).Furthermore,the ability of fungi to form extended mycelial networks makes them well suitable for bioremediation processes.The application of?lamentous fungi can be a promising alternative or a valuable complement in situations of bacterial malfunction(Harms et al.,2011).Arbuscular mycorrhizal(AM) fungi interact with the roots of more than80%of terrestrial plants and are considered functional extensions of plant roots to greatly enlarge the soil volume for nutrient uptake.Plants in symbiosis with AM fungi have the potential to take up metals from an en-larged soil volume.However,members of the Cruciferae,which in-clude major hyperaccumulators,are known as nonmycorrhizal plants and other types of fungal symbionts have not been found in these plant species(Usuki and Narisawa,2007).Compared with AM fungi,endophytic fungi are ubiquitous and comprise a diverse group of fungi(Aly et al.,2010).Fungal endophyte colonization in Pb–Zn polluted plants is moderately abundant and some isolates have a marked adaptation to Pb(II)and Zn(II),which have the po-tential application in phytoremediation(Li et al.,2012).Neverthe-less,most?lamentous fungi are asexual organisms and refractory to transformation and genetic or transgenetic approaches cannot be undertaken to improve their phytoremediation properties (G?hre and Paszkowski,2006).A Trichoderma mutant constructed by restriction enzyme-mediated integration(REMI)has been shown to improve phytoremediation ef?ciency of oilseed rape in cadmium polluted soil(Wang et al.,2009).
Protoplast fusion is an important tool in strain improvement by bringing genetic recombination and developing hybrid strains in ?lamentous fungi(Prabavathy et al.,2006).However,engineered fungal strains constructed by protopalst fusion have not been used in metal pollution control.Therefore,the present study was aimed to isolate protoplasts from endophytic Mucor sp.CBRF59and carry out self-fusion of protoplasts with the objective of enhancing the metal resistance.In addition,phytoremediation of Cd(II)and Pb(II) contaminated soils by rapes,which were inoculated with engi-neered strains and wild strains,were also studied and compared.
2.Materials and methods
2.1.Strain
The endophytic fungus Mucor sp.CBRF59was isolated from the roots of rapes growing in a heavy metal-contaminated soil in our previous study(Deng et al.,2011).The strain were maintained on potato dextrose agar(PDA)and stored at4°C.The strain could grow on the PDA medium with5mM Cd(II)and10mM Pb(II) added.
2.2.Isolation and regeneration of protoplasts
Conidial suspension of Mucor sp.CBRF59was collected by washing the mycelia on slants with sterile water.1mL of conidial suspension(106protoplasts mLà1)was inoculated into a300mL ?ask containing100mL potato dextrose broth(PDB)and shaken at30°C and150rpm for24,36,48,and60h,respectively.The broth was?ltered through?lter paper and the mycelia with differ-ent ages(24,36,48,and60h)were washed with sterile water for three times.The protoplasts of Mucor sp.CBRF59were isolated according the method of Vazquez et al.(1997)with some modi?-cations.About100mg fresh mycelium was immersed in a pre-treatment solution containing0.05M ETDA,2%(v/v)b-Mercaptoethanol in phosphate buffer(pH7),and incubated for 2min at30°C.The mycelia were?ltered and washed three times with a phosphate buffer with0.6M sucrose as an osmotic stabi-lizer.The biomass was then incubated with2mL mixture of2% (w/v)Lywallzyme(Guangdong Institute of Microbiology,China) prepared in phosphate buffer(pH7.0)containing0.6M sucrose as osmotic stabilizer for1–4h.After the incubation,the culture was?ltered through six layers of cheese cloth to remove mycelial fragments.The resultant?ltrate containing protoplasts was washed with osmotic stabilizer(0.6M sucrose)to remove the en-zyme remnants by centrifugation at3500rpm for10min.The sed-imented protoplasts were re-suspended in0.6M sucrose solutions and the number of protoplasts was estimated using the hemacy-tometer and microscope.Protoplasts were regenerated by cultivat-ing on a regeneration medium(the PDA medium containing0.6M sucrose as an osmotic stabilizer).The regeneration frequency(P) was calculated as follows:
P?100%
N c
N p
e1T
where N c is the number of colonies on the regeneration medium, and N p is the number of protoplasts inoculated.
2.3.Self-fusion of protoplasts
The protoplast suspension of CBRF59was inactivated by two different methods(Javadekar et al.,1995;Zhao et al.,2008).The ?rst method is with UV radiation.With5mL of protoplast suspen-sion transferred to an aseptic plate,the plate was exposed to UV radiation for16min at a distance of30cm from a UV lamp with an output power of30W.The second method is with heat:5mL of protoplast suspension in an aseptic centrifuge tube was im-mersed into65°C water for10min.The P of the killed protoplasts by the two methods were detected.Self-fusion of protoplasts of Mucor sp.CBRF59was carried out following Savitha et al.(2010) with some modi?cations.The two types of inactivated protoplasts
42Z.Deng et al./Chemosphere91(2013)41–47
of CBRF59were mixed at the ratio of1:1in0.6M sucrose solutions. The mixed protoplast suspension(about106–107protoplasts mLà1)was centrifuged with3500rpm for10min.The resulting pellets was re-suspended in1mL of the fusant solution(30%of polypropynoglycol6000in a0.05M solution of CaCl2,0.05M gly-cine and0.6M sucrose,pH7)and incubated for20min at30°C. The suspensions were then washed with0.6M sucrose solution and centrifuged at3500rpm for10min.The pellets were har-vested and re-suspended.Then the fused protoplast preparation was transferred to the regeneration medium(PDB containing 0.6M sucrose as an osmotic stabilizer),and kept rest for1d,then cultured on a rotary shaker at60rpm and30°C for2d.The protop-lasts killed only by UV or heating served as the control.
2.4.Selection of self-fusant strains on selective medium
After regeneration in the PDB medium,the culture of the fu-sants was diluted and spread on a selective medium(the PDA med-ium containing3mM Cd(II),and kept at28°C for7d.The regenerated self-fusants of CBRF59were selected based on their fast growth on the selective medium compared to the parent strain.The favorable self-fusants were transferred to new selective plate dishes six times to assess their stability.
2.5.Cd resistance
The stable self-fusants and the parental strain with Cd resis-tance were examined using the PDA medium added with gradually increasing concentrations of Cd(NO3)2(1–30mM).For each con-centration,the strains were incubated at28°C for7d.With an in-creased metal concentration,if no apparent growth of strains was observed on the plates,the metal concentration was considered as the highest concentration tolerated by the tested strains.
2.6.Effects of CBRF59and self-fusant CBRF59T3on the mobility of soil Cd(II)and Pb(II)
The tested soil samples were collected from a heavy metal-con-taminated site in the Dabaoshan mine,Guangdong Province,China. The soil was air-dried and sieved(1.5mm)to remove plant resid-uals,soil macrofauna,and stones.The soil had a pH of2.73,0.53% organic matter,and concentrations of Cd(II),Pb(II),Zn(II)were 19.3,1473,and468mg kgà1,respectively.The soil texture was composed of34%sand,51%silt and15%clay.The effects of CBRF59 and self-fusant CBRF59T3on the mobility of soil Cd(II)and Pb(II) were tested by the method of Deng et al.(2011).
2.7.Effects of CBRF59and self-fusant CBRF59T3on the phytorextraction ef?cacy of Cd(II)-,Pb(II)-contaminated soils Soil samples were collected from the heavy metal-contami-nated sites in Shangba Village,Wengyuan County,Guangdong Province,China.The soil was sieved with a2mm sieve.The soil pH was5.74,organic matter4.2%,and concentrations of Cd(II), Pb(II),Zn(II)were
3.3,862,and373mg kgà1,respectively.The soil texture was composed of44%sand,47%silt and9%clay.The soil sample was supplemented with25mg kgà1of Cd(II)(Cd25), 50mg kgà1of Cd(II)(Cd50),500mg kgà1of Pb(II)(Pb500), 1000mg kgà1of Pb(II)(Pb1000),25mg kgà1of Cd(II)and 500mg kgà1of Pb(II)(Cd25+Pb500),50mg kgà1of Cd(II)and 1000mg kgà1of Pb(II)(Cd50+Pb1000),respectively,by dripping solutions of Cd(NO3)2and Pb(NO3)2.Then the soil samples with the six treatments(the contaminated soils)were left in a green-house for6months for metal stabilization.After metal stabiliza-tion,soil pH values decreased slightly,ranging from5.63to5.72.
Experiments were conducted to investigate the effects of endo-phytic fungi CBRF59and the self-fusant CBRF59T3on the growth and metal uptake of rape(Brassica napus).Seeds of B.napus were sterilized in NaClO solution(6.5%available chlorine)for30min and rinsed several times with sterile distilled water.The seeds were inoculated by soaking in the spore suspension(106spores mLà1)of CBRF59or CBRF59T3for1h.Seeds soaked in sterile water were used as the control.The inoculated and uninoculated seeds were planted in plastic pots(each with400g soil),containing Cd(II)-,Pb(II)-,or Cd(II)+Pb(II)-contaminated soils and the control soil.Ten seeds were sown in each pot and three pots were used per treatment.After the?rst pair of true leaves appeared,three seed-lings were kept in each pot.The plants were grown in a glasshouse under controlled climatic conditions(25°C,with a16h light/8h dark period and60%relative humidity).Plants were watered to maintain soil moisture at about50%of water holding capacity by adding tap water during the experimental period.After45d,the number of survival plants was recorded.Then the plants were re-moved from the pots,shoots and roots were rinsed thoroughly with deionized water.The plant samples were cut into2cm pieces, dried in an oven at105°C overnight,and weighed to determine dry weight biomass.The oven dried samples were grounded to0.5mm using a stainless steel mill.The powder(0.2g)was digested with 10mL of a HNO3–HClO4(4:1,v/v)mixed solution and concentra-tions of Cd(II)and Pb(II)in the shoots and roots were determined with ICP-OES(PerkinElmer Optima,5300DV).
2.8.Recovery of inoculated strains
Recovery of inoculated strains was carried by the method de-scribed by Weyens et al.(2010).The inoculated fungal endophytes were re-isolated from roots of B.napus after45d of growth.The root samples were taken from three plants and pooled together for isolation.The fungal endophytes were isolated as described by Deng et al.(2011).
2.9.Statistical analysis
The translocation factor was de?ned as the ratio of the metal concentration in plant shoots to the metal concentration in plant roots,and used to evaluate the plant’s ability to transfer heavy metals from roots to the harvestable aerial part(Marchiol et al., 2004).Statistical analysis of data was carried out using the SPSS statistical package(version16.0for Windows,SPSS).For the pot experiments,data were represented as mean±standard deviation of three replicates.The data were subjected to ANOVA and treat-ment means were compared by Duncan’s multiple-range test.All analysis was performed at the P60.05level.
3.Results
3.1.Isolation of protoplasts
In present study,the maximum protoplasts released from24, 36,48,and60h old mycelia of Mucor sp.CBRF59were8.7?107, 7.2?107,5.7?107,and4.3?107protoplasts mLà1,respectively. Lywallzyme(2%)reaction mixture with0.6M sucrose at30°C yielded7.5?107,8.8?107,6.6?107,and4.0?107protoplasts mLà1of Mucor sp.CBRF59(with24h mycelial age),respectively, at incubation periods of1,2,3,and4h.The optimal conditions for protoplast release from Mucor sp.CBRF59were with the myce-lial age of24h and incubation period of2h,and8.8?107protop-lasts mLà1was obtained under this conditions.The protoplasts of CBRF59released by this method had a17%P.
Z.Deng et al./Chemosphere91(2013)41–4743
3.2.Selection of self-fusant strains
The inactivated parental protoplasts treated by UV or heat could not be regenerated in the regeneration medium.However,the self-fusants between the protoplasts inactivated by different methods could be regenerated and germinated into mycelia.Based on the mycelial growth,three stable self-fusant strains,which grew mark-edly faster than CBRF59on the PDA medium with3mM Cd(II), were selected and designated as CBRF59T1,CBRF59T2,and CBRF59T3.The self-fusant CBRF59T3showed the highest Cd(II) resistance among the fusants.The highest Cd(II)concentration tol-erated by self-fusant CBRF59T3was25mM,which was4-fold higher than that of parent strain CBRF59.The Pb(II)resistance of CBRF59T3did not show an obvious increase(10mM)compared with the wild type CBRF59.Therefore,the self-fusion CBRF59T3 was selected for further study.
3.3.In?uence of CBRF59and CBRF59T3on the mobility of soil metals
The inoculation of both CBRF59and CBRF59T3signi?cantly im-proved the availability of Cd(II)and Pb(II)in the soil(Table1). Compared with the uninoculation control treatment,the inocula-tion of CBRF59increased the contents of water-soluble Pb(II)and Cd(II)in soils by77%and25%,respectively(P<0.05).The inocula-tion of CBRF59T3increased the contents of water-soluble Pb(II) and Cd(II)in soils by25%and54%,respectively(P<0.05).Com-pared with CBRF59,the inoculation of CBRF59T3further increased the contents of water-soluble Cd(II)in soil by24%(P<0.05).
3.4.Effects of inoculated CBRF59and CBRF59T3on phytoremediation of B.napus
The inoculation of CBRF59and CBRF59T3increased the shoot lengths of B.napus in most of the treatments compared with the uninoculation control.Nevertheless,there was no signi?cant dif-ference between shoot lengths of rape inoculated with CBRF59 and CBRF59T3(Fig.1a).The dry weights of rape inoculated with CBRF59and CBRF59T3were both higher than that of the uninocu-lation rape(Fig.1b).For example,the dry rape weights in the Cd50+Pb1000soil without inoculation,and with CBRF59and CBRF59T3inoculations were0.17,0.23,and0.38g,respectively. Rape inoculated with CBRF59showed higher dry weights than that with CBRF59T3in Cd25,Cd50,and Cd25+Pb500soils.However, inoculation of CBRF59T3increased the dry weight of rape by62% compared with CBRF59in the Cd50+Pb1000soil(P<0.05).
As shown in Table2,in general,Pb(II)concentrations in rape shoots decreased signi?cantly with the CBRF59T3inoculation, compared to the CBRF59inoculation and uninoculated control.
Table1
Content of water-soluble Pb(II)and Cd(II)in soil treated with CBRF59and CBRF59T3,and without the fungus.
Treatment Concentrations of water-soluble Pb(II)
(mg kgà1)Ratio to the total Pb(II)
(%)
Concentrations of water-soluble Cd(II)
(mg kgà1)
Ratio to the total Cd(II)
(%)
Control(water+soil)0.65±0.03*c**0.040.44±0.02c 2.3 CBRF59+soil 1.15±0.06a0.080.55±0.03b 2.9 CBRF59T3+soil0.81±0.06b0.050.68±0.05a 3.5
*The values presented in columns2and4are mean±standard deviation(n=3).
**Different small letters indicate that comparison differences are signi?cant(ANOVA,P<0.05).
44Z.Deng et al./Chemosphere91(2013)41–47
However,Pb(II)concentrations in rape roots of the CBRF59T3inoc-ulation,CBRF59inoculation,and uninoculated control were not signi?cantly https://www.sodocs.net/doc/091951219.html,pared to inoculation of CBRF59,inocu-lation of CBRF59T3increased Cd(II)concentrations in rape shoots by52%,187%,129%,35%in the Cd25,Cd50,Cd25+Pb500,and Cd50+Pb1000soils,https://www.sodocs.net/doc/091951219.html,pared with the uninocula-tion control,inoculations of CBRF59T3and CBRF59resulted in de-clined Cd(II)concentrations in rape roots in most cases.However, there was no signi?cant difference between the Cd(II)concentra-tions in rape roots inoculated with CBRF59T3and CBRF59.The inoculation of CBRF59T3enhanced the Cd(II)translocation from roots to https://www.sodocs.net/doc/091951219.html,pared to the uninoculated control,inoculation of CBRF59T3increased the translocation factors of Cd(II)(TF Cd)in rape by111%,181%,136%,and64%in Cd25,Cd50,Cd25+Pb500,and Cd50+Pb1000soils,https://www.sodocs.net/doc/091951219.html,pared to inoculation of CBRF59,inoculation of CBRF59T3increased TF Cd values by78%, 127%,111%,and47%in Cd25,Cd50,Cd25+Pb500,and Cd50+Pb1000soils,respectively.However,the translocation fac-tors of Pb(II)declined when the rape was inoculated with CBRF59 or CBRF59T3(P<0.05).
Compared to the uninoculated control,the inoculation of CBRF59improved the extraction amount of Cd(II)by rape shoots in the treatments except Pb500and Cd50+Pb1000(Fig.2a).Com-pared to CBRF59,CBRF59T3markedly increased the extraction amount of Cd(II)in the tested soils amended with Cd(II).In Cd25, Cd50,Cd25+Pb500,and Cd50+Pb1000soils,the extracted amount of Cd(II)by the rape inoculated with CBRF59T3were 31%,92%,37%,and119%,respectively,higher than those with
Table2
Effect of CBRF59and CBRF59T3on the concentrations and translocation of Cd and Pb in rape.
Soil treatments Uninoculated control rape Rape inoculated with CBRF59Rape inoculated with CBRF59T3
CK C S
Pb
A25±2B a**23±2a17±1b
C R Pb220±13a232±9a237±52a
TF Pb0.12±0.01a0.10±0.01a0.07±0.01b
C S Cd6±0.7a5±0.4a6±0.3a
C R Cd36±8a28±5a23±2a
TF Cd0.17±0.03b0.19±0.03ab0.26±0.04a
CK with25mg kgà1Cd(II)C S
Pb
19±5a21±3a5±1b
C R Pb232±38a168±20b148±6b
TF Pb0.08±0.03b0.13±0.00a0.04±0.01c
C S Cd79±14a50±3b76±2a
C R Cd299±51a155±7b136±13b
TF Cd0.27±0.06b0.32±0.02b0.57±0.05a
CK with50mg kgà1Cd(II)C S
Pb
17±0.3a13±1b8±1c
C R Pb237±35a239±28a173±8b
TF Pb0.07±0.01a0.06±0.01ab0.05±0.01b
C S Cd106±6b78±5c224±13a
C R Cd482±21a304±13c381±25b
TF Cd0.21±0.04b0.26±0.03b0.59±0.03a
CK with500mg kgà1Pb(II)C S
Pb
25±6a29±4a11±2b
C R
Pb
321±40ab442±100a228±19b
TF Pb0.08±0.01a0.07±0.01a0.05±0.01a
C S
Cd
11±1a7±0.7b8±0.5b
C R
Cd
39±3a27±2b22±1c
TF Cd0.29±0.06b0.28±0.04b0.34±0.01a
CK with1000mg kgà1Pb(II)C S
Pb
37±7a19±2b22±0.4b
C R
Pb
474±91a460±17a530±18a
TF Pb0.08±0.03a0.04±0.01b0.04±0.00b
C S Cd5±0.2a3±0.4b5±0.3a
C R Cd42±12a20±2b23±3b
TF Cd0.12±0.04b0.17±0.02ab0.20±0.02a
CK with25mg kgà1Cd(II)+500mg kgà1Pb(II)C S
Pb
20±1a12±1b9±0.5c
C R Pb280±5a249±13a277±17a
TF Pb0.07±0.00a0.05±0.00b0.03±0.00c
C S Cd75±10b49±6c112±7a
C R Cd288±56a176±21b192±9b
TF Cd0.25±0.00b0.28±0.02b0.59±0.05a
CK with50mg kgà1Cd(II)+1000mg kgà1Pb(II)C S
Pb
21±2a25±3a12±1b
C R Pb540±57a605±22a535±27a
TF Pb0.04±0.01a0.04±0.00a0.02±0.00b
C S Cd131±13a104±7b140±5a
C R Cd772±84a555±34b503±33b
TF Cd0.17±0.02b0.19±0.02b0.28±0.02a
CK represents the control soil without addition of metals.
A C S
Pb
(or C S Cd)represents the concentration of Pb(or Cd)in shoots of rape(mg kgà1dry biomass);C R Pb(or C R Cd)represents the concentration of Pb(or Cd)in roots of rape (mg kgà1dry biomass);TF Pb(or TF Cd)represents the translocation factor of Pb(or Cd)in rape.
B The values are mean±standard deviation(n=3).
**Different small letters indicate that the multiple comparison differences are signi?cant with different fungi treatments and the same soils(P<0.05).
Z.Deng et al./Chemosphere91(2013)41–4745
CBRF59.The inoculation of CBRF59T3and the uninoculated control resulted in similar amount of Pb(II)extracted by rape,while the Pb(II)extraction ef?cacy by rape with CBRF59inoculation was higher(Fig.2b).
3.5.Recovery of inoculated fungal endophytes
After45d of growth,the roots of rape were harvested.The cul-tivable endophytic fungi were isolated from the roots.The Cd-resistant endophytic Mucor sp.CBRF59and self-fusant CBRF59T3 could be re-isolated from the roots of inoculated rape and the number of CFU that could be re-isolated tended to increase when rape were exposed to Cd-contaminated soils.
4.Discussion
The Cd(II)concentration tolerated by mutant P6constructed by REMI increased2-fold than that of wild type strains,the amount of Cd(II)in shoots of mutant P6treated oilseed rape increased by23% and38%per pot compared with the wild type Trichoderma treat-ment in20and50mg Cd(II)kgà1soil(Wang et al.,2009).In this study,the mutant CBRF59T3constructed by self-fusion of proto-plast of CBRF59tolerated4-fold higher Cd(II)concentration (25mM)than that of wild type https://www.sodocs.net/doc/091951219.html,pared with CBRF59, the Cd(II)concentrations in shoots of rape inoculated with CBRF59T3increased by187%and the Cd(II)amount extracted by rape increased by92%,the value of TF Cd increased by127%in the Cd50soil.In addition,the mutant CBRF59T3could increase the Cd(II)extraction ef?cacy in Cd(II)-+Pb(II)-contaminated soils compared with wild type CBRF59.Therefore,the mutant CBRF59T3 constructed by self-fusion of protoplast should be more advanta-geous than that of mutant P6constructed by REMI.
The REMI is a tagging mutagenesis procedure in fungi.Although mutagenic by REMI offers distinct advantages over the conven-tional mutagenesis in?lamentous fungi,in most systems,only 50%of the genes appear to be tagged and to determine linkage may require a substantial amount of additional work such as ef?-cient transposon mutagenesis,while reliable heterologous trans-poson tagging techniques are not yet available for?lamentous fungi(Kahmann and Basse,1999).Furthermore,the REMI proce-dure requires the vector molecule with selectable marker and the fungal protoplasts were needed for transformation(Wang et al., 2009).Compared to the REMI methods,protoplast fusion used in this study is more advantageous tool in strain improvement for bringing genetic recombination and developing hybrid strains in ?lamentous fungi(Prabavathy et al.,2006;Gunashree and Venk-ateswaran,2010).In fungi that lack natural mechanism for recom-bination of genetic material,protoplast fusion provides a method to facilitate heterokaryon formation,which were potentially lead-ing to fusion of vegetative nuclei and mitotic recombination even across species and genus barrier that results in the development of interspeci?c and intergenetic hybrids(Savitha et al.,2010).Pro-toplast fusion is a novel approach,through which potential strains with desirable properties could be obtained with minimal distur-bance in the physiology.The method allows to combine entire chromosomes that are separated by species and genus barriers, allowing introduction of novel characteristics into strains(Vazquez et al.,1997).The characteristics of mutant CBRF59T3constructed by self-fusion of CBRF59protoplast also demonstrated the advan-tages.The mutant could increase the Cd(II)extraction ef?cacy in Cd(II)-or Cd(II)-+Pb(II)-contaminated soils compared with wild type CBRF59and the uninoculated control.Nevertheless,the mu-tant did not show signi?cant higher Pb(II)extraction ef?cacy than the wild type CBRF59.The result might be attributable to that the resistance of CBRF59T3to Pb(II)did not increase more signi?cantly than that of CBRF59.In this study,the dry biomass of rape in soils with higher Pb(II)concentration were higher than that in the soils with lower Pb(II)concentration.The similar results were obtained from rape in other studies(Cao et al.,2007;Wang et al.,2009).It may be attributable to that the metals added within a certain range in soils could enhance the bioavailability of other essential metal
46Z.Deng et al./Chemosphere91(2013)41–47
ions,which resulted in promoting the plant growth(Patra et al., 1994;Gadd,2000;Cao et al.,2007).
To facilitate screening and identi?cation of the recombinant,ge-netic markers(e.g.,auxotrophic strains)are used when hybrids are constructed.However,the genetic markers affect the physiology and metabolism of strains and lead to reduced performance in function working(Gong et al.,2009).In the study,the protoplasts were pretreated with UV or heat to achieve inactivation.The lethal injury inactivated with the different methods could be comple-mented and thus the fusion body with physiological activities could be screened(Gong et al.,2009).The inactivation of the parent protoplasts can avoid the numerous processes of genetic marking and increase the screening ef?ciency of the recombinants.
The fungal detoxifying function and environmental tolerance are a particularly complex and poorly understood phenotype and many of the tolerance phenotypes are polygenic that involve dis-tributed genes in the genome(Gong et al.,2009).Therefore,it is challenging to develop engineered fungi by recombinant of the me-tal resisting genes.Our results demonstrated that protoplast fusion is a feasible and ef?cient method to improve stress tolerance of uncharacterized fungi for metal pollution control.To our knowl-edge,this study is the?rst to demonstrate that the mutants con-structed by protoplast fusion signi?cantly increase phytoextraction ef?ciency of metal-contaminated soils.
Acknowledgments
This work was partly supported by grants from the Chinese Na-tional Natural Science Foundation(Nos.51039007and51179212) and the Fundamental Research Funds for the Central Universities. References
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简单三角恒等变换典型例题
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如何写先进个人事迹
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空间向量及其运算
§8.5 空间向量及其运算 1． 空间向量的概念 (1)定义：空间中既有大小又有方向的量叫作空间向量． (2)向量的夹角：过空间任意一点O 作向量a ，b 的相等向量OA →和OB → ，则∠AOB 叫作向量a ，b 的夹角，记作〈a ，b 〉，0≤〈a ，b 〉≤π. 2． 共线向量定理和空间向量基本定理 (1)共线向量定理 对空间任意两个向量a ，b (b ≠0)，a ∥b 的充要条件是存在实数λ，使得a ＝λb . (2)空间向量基本定理 如果向量e 1，e 2，e 3是空间三个不共面的向量，a 是空间任一向量，那么存在唯一一组实数λ1，λ2，λ3使得a ＝λ1e 1＋λ2e 2＋λ3e 3，其中e 1，e 2，e 3叫作空间的一个基底． 3． 空间向量的数量积及运算律 (1)定义 空间两个向量a 和b 的数量积是一个数，等于|a ||b |cos 〈a ，b 〉，记作a ·b . (2)空间向量数量积的运算律 ①结合律：(λa )·b ＝λ(a·b )； ②交换律：a·b ＝b·a ； ③分配律：a·(b ＋c )＝a·b ＋a·c . 4． 空间向量的坐标表示及应用 (1)数量积的坐标运算 设a ＝(a 1，a 2，a 3)，b ＝(b 1，b 2，b 3)， 则a·b ＝a 1b 1＋a 2b 2＋a 3b 3. (2)共线与垂直的坐标表示 设a ＝(a 1，a 2，a 3)，b ＝(b 1，b 2，b 3)， 则a ∥b ?a ＝λb ?a 1＝λb 1，a 2＝λb 2，a 3＝λb 3 (λ∈R )， a ⊥b ?a·b ＝0?a 1b 1＋a 2b 2＋a 3b 3＝0(a ，b 均为非零向量)． (3)模、夹角公式 设a ＝(a 1，a 2，a 3)，b ＝(b 1，b 2，b 3)， 则|a |＝a·a ＝a 21＋a 22＋a 23，
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最新小学生个人读书事迹简介怎么写800字
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三、生活朴素，乐于助人 平时重视与同事间的关系，主动与同事打成一片，善于发现他人的难处，及时妥善地给予帮助。在其它同志遇到困难时，积极主动伸出援助之手，尽自己最大努力帮助有需要的人。养成了批评与自我批评的优良作风，时常反省自己的工作，学习和生活。不但能够真诚的指出同事的缺点，也能够正确的对待他人的批评和意见。面对误解，总是一笑而过，不会因为误解和批评而耿耿于怀，而是诚恳的接受，从而不断的提高自己。在生活上勤俭节朴，不铺张浪费。 身为一名老党员，我感到责任重大，应该做出表率，挤出更多的时间来投入到**党总支的工作中，不找借口，不讲条件，不畏困难，将总支建设摆在更重要的位置，解开工作中的思想疙瘩，为攻坚克难铺平道路，以支部为纽带，像战友一样团结，像家庭一样维系，像亲人一样关怀，践行入党誓言。把握机遇，迎接挑战，不负初心。
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