!两个抗虫转基因棉花品种(陆地棉)单叶光合特性对光的反应——东北大学2009

PHOTOSYNTHETICA 47 (3): 399-408, 2009399

Single leaves photosynthetic characteristics of two insect-resistant

transgenic cotton (Gossypium hirsutum L.) varieties in response to light

C.X. SUN *,+, H. QI **, J.J. HAO *, L. MIAO *, J. WANG *, Y. WANG *, M. LIU **, and L.J. CHEN ***

Department of Biology, Science College, Northeastern University, P.O. Box 325, 110004, Shenyang, P. R. China * Agronomy College, Agricultural University of Shenyang, 110016 Shenyang, P. R. China ** Institute of Applied Ecology, Chinese Academy of Sciences, 110006 Shenyang, P. R. China ***

Abstract

How the photosynthetic characteristics of insect-resistant transgenic cotton (Gossypium hirsutum L.) respond to light or whether this genetic transformation could result in unintended effects on their photosynthetic and physiological processes is not well known. Two experiments were conducted to investigate the shapes of net photosynthetic rate (P N ), stomatal conductance (g s ), apparent light use efficiency (LUE app ) and water use efficiency (WUE) light-response curves for single leaves of Bt (Bacillus thuringiensis ) and Bt +CpTI (cowpea trypsin inhibitor) transgenic cotton plants and their non-transgenic counterparts, respectively. Results showed that the significant difference in response of P N and WUE to light between transgenic cotton and non-transgenic cotton occured but not always throughout the growing season or in different experiments or for all transgenic cotton lines. It was highly dependent on growth stage, culture condition and variety, but no obvious difference between any transgenic cotton and non-transgenic cotton in the shapes of g s and LUE app light-response curves was observed in two experiments at different growth stages. In the field experiments, transgenic Bt +CpTI cotton was less sensitive to response of P N to high irradiance at the boll-opening stage. In pot experiments, WUE light-response curves of both Bt transgenic cotton and Bt +CpTI transgenic cotton progressively decreased whereas non-transgenic cotton slowly reached a maximum at high irradiance at boll-opening stage. We supposed that culture environment could affect the photosynthesis of transgenic cotton both directly and indirectly through influencing either foreign genes expression or growth and physiological processes.

Additional key words : apparent light use efficiency; Bacillus thuringiensis ; light-response curve; net photosynthetic rate; stomatal conductance; transgenic cotton; trypsin inhibitor; water use efficiency.

Introduction

The production of insect-resistant transgenic cotton is supposed to bring significant economic benefits and result in good ecological benefits (Qaim and Zilberman 2003). Since 1997, China has formally approved com-mercial production of transgenic cotton, and in 2007, the total planting area of insect-resistant transgenic cotton reached 380 million hectares, accounting for 69 % of the total planting area of cotton in our country (Mo 2007). Photosynthesis is the physiological basis of crop growth and production, and a determining factor of crop yield. On one hand, stomata are the joining point between carbon and water circles in ecological systems, on the other hand, stomata are the pathway that permits the entrance of CO 2 and simultaneous loss of water vapor and then controls the balance between H 2O lost and CO 2 assimilated (Wullschleger and Oosterhuis 1989, Yu et al . 2001). Studies have been conducted looking at the response of transgenic insect-resistant cotton in terms of gas exchange properties. Dong et al. (2006) reported that three Bt cotton varieties had showed different curvilinear changes in the diurnal course of leaf photosynthetic rate. Hebbar et al. (2007) pointed out that the stomatal

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Received 27 February 2009, accepted 13 August 2009. +

Author for correspondence; fax: +86-24-23128449 , e-mail: suncaixia@https://www.sodocs.net/doc/0916210153.html,

Abbreviations : α – the apparent quantum yield for CO 2 assimilation; Bt – Bacillus thuringiensis ; C i – intercellular CO 2 concentration; CPTI – cowpea trypsin inhibitor; E m – the rate of transpiration; g s – stomatal conductance; LUE app – apparent light use efficiency; P max,i – the maximum net photosynthetic rate at 400 μmol mol –1 of CO 2; P N – the net photosynthetic rate; PPFD – photosynthetic photon flux density; R D – the apparent dark respiration rate; WUE – water use efficiency.

Acknowledgements : The study was financially supported in part by Initial Funding for Ph D of Liaoning Province (No. 200412), Programs for Science and Technology Development of Liaoning Province (No. 2004201003) and National Natural Science Foundation of China (No. 40101016), P.R. of China. We gratefully acknowledge Dr. Wendy Harwood from Crop Genetics Department, John Innes Centre, UK, for language correction.

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conductance rates of transpiration and photosynthesis did not differ significantly between Bt and non-Bt counter-parts up to 80 days after sowing. Our former results also showed that the changes in g s , transpiration rate (E m ) and intercellular CO 2 concentration (C i ) in the leaves of Bt and Bt +CpTI transgenic cotton were not significantly different to non-transgenic cotton. However, the differen-ce of P N between Bt transgenic cotton and non-Bt cotton was significant at the seedling stage (Sun et al . 2007). Light plays a key role in photosynthesis and productivity of crops by providing the energy needed for assimilatory power, activating enzymes concerned with photosyn-thesis, promoting the opening of stomata, and regulating the development of the photosynthetic apparatus (Xu 2002). Among environmental factors, photosynthetic photon flux density (PPFD) is particularly subjected to a rapid and marked fluctuation in the field. This may require a rapid and efficient response of plant physio-logical processes to light, and thus limitation of these processes by light could potentially be minimized (Yu et al . 2001). However, how these physiological processes or characteristics of insect-resistant transgenic cotton response to light, are not well known.

Therefore, the objectives of the present study were to investigate responses of P N , g s , LUE app and WUE of Bt and Bt +CpTI transgenic cotton to light, and to describe any unintended effects of transgene insertion on the transgenic cotton in photosynthetic physiological terms. This information would be valuable in discussion on the use of transgenic cotton.

Materials and methods

Cotton culture : The pot and field experiments were conducted at the Experimental Station of Shenyang Agricultural University (SAU), Shenyang (123°4′E, 41°8′N), Liaoning. Two types of indigenous Chinese commercial insect-resistant transgenic cotton including the Bt transgenic cotton Z30, the Bt +CpTI transgenic cotton SGK321, and their non-transgenic parental counterparts Z16 and SY321 were used in these experiments, respectively. Acid-delinted seeds of each variety were kindly provided by the Germ Plasma Resources Centre, Institute of Cotton, Chinese Academy of Agricultural Sciences (Anyang, Henan).

Cotton seeds were sown in pots containing 15 kg brunisolic soil obtained from the plough layer in the field at the Experimental Station of SAU in an outside growing area at the Experimental Station of SAU in mid May 2006. The soil in two experiments is a brunisolic soil having pH 5.72, organic matter 2.52 g kg –1, total N 1.22 g kg –1, total P (P 2O 5) 1.12 g kg –1, total K (K 2O) 24.24 g kg –1. Six pots were used for each variety and the plant population was thinned with three plants maintained per pot two weeks after emergence. Water stress was mini-mized with timely irrigation and insecticides were applied as needed during the season.

In 2007, the field experiments were arranged in a randomized complete block design with three replica-tions. Each plot was formed by five rows with row length of 8 m and plant population density was 4.5 plants m –2. Cotton seedlings were transplanted in early May.

Fertilizer consisted of 225–82.5–187.5 kg ha –1 of

N–P 2O 5–K 2O incorporated before planting. Side-dressing with 90 kg(N) ha –1 was conducted 10 weeks after planting. Furrow irrigation provided a well-watered environment and insecticides were applied as needed during the season. Intensive management in cotton fields was carried out according to local agronomic practices unless otherwise indicated.

Photosynthetic characteristics measurements : P N , g s , and E m of single leaves were measured on the second young fully mature leaf on the main stem at squaring and boll-opening stages in 2006 (a pot experiment) and 2007 (a field experiment) with a portable photosynthesis system LI-6400 (LI-COR , Lincoln, NE, USA). During the measurements of light response curves of photosynthetic characteristics, PPFD was 0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and

2000 μmol(photon) m –2 s –1, adjusted automatically by

a red:blue light source (LI-6400-02BL ED; LI-COR ). The leaves were held at each PPFD for a minimum of 30 min prior to determination to allow equilibration of the photosynthetic system to that PPFD. The temperature, relative air humidity and CO 2 concentration in leaf chamber were kept at 30 °C, 60 % and 400 μmol mol –1, respectively. All readings were made between 9:00 and 11:00 hours on cloudless days.

Model fitting and data analysis : The light response curves of P N were fitted to a Michaelis-Menten model based on measurement of P N and PPFD (Thornley 1976).

D i

max,i max,N PPFD PPFD R P P P ?+αα=

,

where α is the apparent quantum yield for CO 2 assimilation, P max,i is the maximum net photosynthetic rate at 400 μmol mol –1 of CO 2, and R D is the apparent dark respiration rate. These parameters were estimated using Nonlinear Regression in SPSS 11.0 based on Michaelis-Menten model.

LUE app was calculated by using the equation:

PPFD

LUE N

app P =

(Long et al . 1993).

WUE was calculated by using the equation:

PHOTOSYNTHETIC CHARACTERISTICS OF INSECT-RESISTANT TRANSGENIC COTTON IN RESPONSE TO LIGHT 401

m

N

WUE E P =

(Nijs et al . 1997). Data were statistically analyzed by the ANOVA procedures in SPSS 11.0 (Chicago, USA). All measure-ments were recorded from six replications at each sampling date.

Results

P N : A non-rectangular hyperbolic curve has been widely used to describe photosynthetic light-response curves. P N of all test cotton varieties during their whole growth season fitted the non-rectangular hyperbolic equation well. Parameters α, P max,I and R D , defining the fitted curves, were summarized in Table 1.

Fig. 1 shows the light-response curve for P N , con-structed using the estimated value calculated by the

parameters in Table 1. On the whole, at low irradiance (below 200 μmol m –2 s –1), all transgenic insect-resistant cotton had similar shapes of photosynthetic light-response curve as their non-transgenic counterparts. However, the difference between transgenic insect-resistant cotton and non-transgenic cotton in the shape of photosynthetic light-response curve broadened with the increase in irradiance (Fig. 1).

Table 1. Parameters of photosynthesis in response to light intensity between two transgenic insect-resistant cotton (SGK321, Z30) and their non-transgenic counterparts (SY321, Z16) at squaring and boll opening stages in 2006 (pot experiment) and 2007 (field experiment). Values in each row followed by the same letters are not significantly different (p <0.05) according to Duncan’s multiple range test. α – the apparent quantum yield; P max,i – the maximum net photosynthetic rate; R D – the apparent dark respiration rate. Means (n = 6).

Variety

Year Stage Parameter

SY321 SGK321 Z16

Z30

α 0.067a 0.060a 0.068a 0.070a

P max,i [μmol CO 2 m –2 s –1

] 36ab 33a 42b 34a R D [μmol CO 2 m –2 s –1 ] 3.0a 3.2a 4.0b 4.3b Squaring stage

r 2

0.9979 0.9975 0.9983 0.9984 α

0.059a 0.063a 0.089a 0.069a P max,i [μmol CO 2 m –2 s –1

] 22a 18a 17a 16a

R D [μmol CO 2 m –2 s –1

] 1.8bc 1.4ab 2.0c 1.2a 2006

Boll opening stage r 2

0.9984 0.9982 0.9731 0.9963 α 0.080a 0.071a 0.072a 0.072a

P max,i [μmol CO 2 m –2 s –1

] 30a 23a 35a 33a

R D [μmol CO 2 m –2 s –1

] 2.6a 2.2a 3.1a 2.5a Squaring stage

r 2

0.9894 0.9946 0.9951 0.9978 α 0.075a 0.070a 0.086a 0.070a

P max,i [μmol CO 2 m –2 s –1

] 35b 23a 28ab 21a

R D [μmol CO 2 m –2 s –1 ]

3.0b 1.8a 2.7ab 2.0ab 2007

Boll opening stage r 2

0.9984 0.9982 0.9731 0.9963

In the pot experiments, at squaring stage, P N of two

varieties of transgenic insect-resistant cotton increased over the entire course of the light-response curve, and the difference in P N between transgenic Bt cotton Z30 and its non-transgenic counterpart Z16 was more distinct than that of transgenic Bt +CpTI cotton SGK321 compared to non-transgenic counterpart SY321 (Fig. 1A ). On the other hand, parameters α and R D did not significantly vary between any transgenic cotton and their non-transgenic counterpart, however, P max,i of Bt cotton Z30 decreased 19 % more than its non-transgenic counterpart Z16 and the difference was significant. Thus, the difference between transgenic Bt cotton Z30 and non-transgenic cotton Z16 in the response of P N to light at the squaring stage in the pot experiments was due to a change in P max,i (p <0.05) but not in the parameter α and R D , implying a change in high light use efficiency (LUE) (Stirling et al . 1993). The difference between transgenic cotton and non-transgenic cotton in the shape of the photosynthetic light-response curve at boll-opening stage was less obvious than that at squaring stage, especially for transgenic Bt cotton Z30 with a similar shape of curve as non-transgenic cotton Z16 at high irradiance range (Fig. 1B ). Moreover, parameters α and P max,i were not significantly different between any transgenic cotton and their non-transgenic counterparts at boll-opening stage, but R D of Bt cotton Z30 decreased 40 % more than its non-trans-genic counterpart Z16 and the difference was significant in the pot experiments. R D change was usually related to changes in C i , enzymatic activity, dark CO 2 fixation rate,

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Fig. 1. Response of P N to light intensity of the second young fully mature leaves on main stem between two transgenic insect-resistant cotton (□ SGK321, ○ Z30) and their non-transgenic counterparts (■ SY321, ● Z16) at squaring (A ,C ) and boll opening stages (B ,D ) in 2006 (A ,B , pot experiment) and 2007 (C ,D , field experiment). Each data point represents estimated value using Michaelis-Menten model, in which adopted value of α, P max,i and R D are shown in Table 1, respectively. Means (n = 6).

or nonstructural saccharides (Shaish et al . 1989, Qiao et al. 2007). Low R D underlined the low metabolic activity of transgenic Bt cotton Z30 during the later growing season compared with non-transgenic cotton Z16 (Gratani et al . 2007).

In the field experiments, P N of transgenic Bt +CpTI cotton SGK321 increased in a distinctly different way over the entire course of the light response curve compared to non-transgenic SY321 at the boll-opening stage, but there were no statistically significant differen-ces in parameters between any transgenic insect-resistant cotton and their non-transgenic counterpart at the squaring stage (Fig. 1C ). Likewise, no statistically significant differences in any parameters of transgenic Bt cotton Z30 were observed compared to non-transgenic cotton Z16 at the boll-opening stage. However, both P max,i and R D of transgenic Bt +CpTI cotton SGK321 were decreased significantly accompanied by the inhibition of P N under high irradiance conditions while non-transgenic cotton SY321 could maintain a fairly high rate of photosynthesis (Fig. 1D ). In this case, in the lower PPFD range (below 200 μmol m –2 s –1), light plays a dominant limiting role in photosynthesis, apparent quantum yield of SGK321 observed from the light response curve did not

change significantly (Table 1). In the period of P N curvilinear increase, SGK321 exhibited lower P N than SY321 caused possibly by either poor capacity to activate Rubisco that is a key enzyme in the process of carbon fixation, or poor capacity to provide energy to form assimilatory power, or poor capacity to regulate the stomata opening, meaning inadequate absorbtion of CO 2 (Xu 2002). P max,i of transgenic Bt +CpTI cotton SGK321 decreased significantly in comparison with SY321 indicating that photoinhibition occurred at exposure to high irradiance caused by excessive light energy absorption (?gren and Evans 1993). The term photo-inhibition has been used to describe light induced reduction of photosynthesis arising from either damage to the D1 protein of PSII reaction centers or increases in non-photochemical quenching of PSII excitation energy (Bradbury and Baker 1986). Chow (1994) has pointed out that plants could protect their photosynthetic apparatus from photodamage through several pathways by thermal dissipation. We deduced that decreases in the efficiency of electron transport and the content of photosynthetic key enzymes such as Rubisco could result in a reduction in photosynthesis in SGK321. On the other hand, decreased operation of protective thermal dissipation or

PHOTOSYNTHETIC CHARACTERISTICS OF INSECT-RESISTANT TRANSGENIC COTTON IN RESPONSE TO LIGHT

403

Fig. 2. Comparison of LUE versus light intensity curves of the second young fully mature leaves on main stem of two trans-genic insect-resistant cotton (□ SGK321, ○ Z30) and their non-transgenic counterparts (■ SY321, ● Z16) at squaring (A ,B ,E ,F ) and boll-opening stages (C ,D ,G ,H ) in 2006 (A ,B ,C ,D , pot experiment) and 2007 (E ,F ,G ,H , field experiment). Means ±SD are shown (n = 6).

limitation of the removal of storage matter caused by a significant decline in R D also might result in the photoinhibition of SGK321 at high irradiance (Niyogi 1999). Apparently, transgenic cotton SGK321 could not response to high light conditions rapidly and efficiently in field experiments.

LUE app : The shapes of the light-response curves of LUE app for cotton studied in our research all exhibited two distinct phases; a rapid increase to maximum at low irradiance from 100 μmol m –2 s –1 to 400 μmol m –2 s –1, and a period of linear decline to negligible LUE app at high irradiance (Fig. 2). In both pot and field experiments,

C.X. SUN et al.

404 Fig. 3. Comparison of g s versus light intensity curves of the second young fully mature leaves on main stem of two trans-genic insect-resistant cotton (□ SGK321,○ Z30) and their non-transgenic counter-parts (■ SY321, ● Z16) at squaring (A,B,E,F) and boll-opening stages (C,D,G,H) in 2006 (A,B,C,D, pot experiment) and 2007 (E,F,G,H, field experiment). Means ±SD are shown (n = 6).

LUE app of transgenic insect-resistant cotton reached a maximum with values slightly lower than, or similar to, the non-transgenic counterpart at a certain irradiance, and then declined much quickly than in the non-transgenic counterpart except for transgenic Bt cotton Z30at squaring stage in the field experiments. In this case, no obvious difference between Z30 and Z16 in LUE app was observed (Fig. 2F).

PHOTOSYNTHETIC CHARACTERISTICS OF INSECT-RESISTANT TRANSGENIC COTTON IN RESPONSE TO LIGHT

405

Fig. 4. Comparison of WUE versus light intensity curves of the second young fully mature leaves on main stem of two trans-genic insect-resistant cotton (□ SGK321, ○ Z30) and their non-transgenic counterparts (■ SY321, ● Z16) at squaring (A ,B ,E ,F ) and boll-opening stages (C ,D ,G ,H ) in 2006 (A ,B ,C ,D , pot experiment) and 2007 (E ,F ,G ,H , field experiment). Means ±SD are shown (n = 6).

g s : Although the data were somewhat scattered, results indicated that g s of all cotton studied in this paper markedly increased with light over the entire course of the light response curve (Fig. 3). The increases in g s of transgenic cotton were slighter than their non-transgenic counterpart, however, g s of transgenic cotton Z30 at boll-opening stage exhibited the same shape of curves as its non-transgenic cotton Z16 both in pot and field experiments (Fig. 3D , H ).

C.X. SUN et al.406

WUE : Water use efficiency is an often used parameter which relates gas exchange fluxes of carbon dioxide and water vapor and quantifies the total amount of CO 2 fixed per unit water lost (Wullschleger and Oosterhuis 1989). Overall, in low irradiance ranges from starting point to about 1000 μmol m –2 s –1 both in pot and field experi-ments, WUE of all cotton progressively increased with light to a maximum, whereas at high irradiance most cotton remained steadily at the maximum (Fig. 4).

In the pot experiments, at the squaring stage, the shape of light-response curves of WUE of two varieties of transgenic cotton were similar to their non-transgenic counterpart respectively (Fig. 4A ,B ). However, WUE of both transgenic Bt cotton Z30 and transgenic Bt +CpTI cotton SGK321 decreased slowly rather than remaining steady after reaching saturation at high irradiance at the boll-opening stage (Fig. 4C ,D ). These changes in WUE with PPFD could not be explained solely by variations in g s since increases in g s with PPFD were almost similar for all cotton varieties (Fig. 3). WUE of plants depends on photosynthesis coupled with transpiration through regulation of stomata opening. However, differing from transpiration, photosynthesis is also an intrinsic biochemical reaction and is inhibited by feedback of photosynthetic products and also reflects the heterogeneous character of diffusivity of CO 2 and H 2O (Yu et al . 2001). Since light probably has a more direct limit on the photochemical processes of P N than on the physical processes controlling transpiration, WUE can be expected to rise with increases in PPFD at low irradiance (Wullschleger and Oosterhuis 1989). After incubation under low light the activation of photosynthetic enzymes is faster than simultaneous opening of stomata (Xu 2002). In the field experiments, no obvious differences in WUE between any transgenic cotton and their non-transgenic counterpart were seen in the low irradiance range. On the other hand, the difference between trans-genic insect-resistant cotton and non-transgenic cotton in the shape of WUE light-response curve broadened with the increase in irradiance (Fig. 4E ,F ,G ,H ).

Discussion

There has been a significant debate concerning the potential unintended effects of insertion of the foreign gene into transgenic crops (Conner and Jacobs 2000, Saxena and Stotzky 2001). Although the methods used to produce transgenic crops are being continually improved, it is not possible at present to control the exact stability, integration and expression of the inserted gene into the plant genomes, that is, it may alter the plant charac-teristics in physiology, anatomy and metabolism as a result of secondary or pleiotropic effects of the transgene expression and insertion (Cellini et al . 2004, Shrawat and L?rz 2006).

Our present data indicate that substantial differences did occur in the shape of P N and WUE light-response curves between transgenic cotton and non-transgenic parental counterparts both grown in the field and pots, respectively (Table 1, Figs. 1A,D ; 4C ,D ). However, the change in P N with respect to PPFD suggested that leaves of transgenic cotton exposed to saturating light intensities were less capable of assimilating of CO 2 compared to non-transgenic cotton leaves either due to possible photoinhibition or other unintended effects of transgene insertion or the transformation process which were not studied in this paper (Cellini et al . 2004). It was even as Ashok and Horst (2006) reviewed that many factors could contribute to variation in transgene expression including tissue culture-induced variation or chimerism in the primary integration site (position effects), transgene copy number (dosage effects), transgene mutation and epigenetic gene silencing.

Wells (1988) has presented information that cotton leaves, which emerged during vegetative growth,

had higher P N levels than those presented in leaves,

which emerged during periods of fruit development.

Wullschleger and Oosterhuis (1990) have also pointed out that the response of P N and g s to incident PPFD conditions during canopy development was highly age-dependent. There were substantial adjustments in leaf physiology and morphology in response to the ambient light environment and this ability of leaves to alter the photosynthetic apparatus has also been recognized to depend closely on the developmental stage of the cotton tissue (Sassenrath-Cole et al . 1996, Dong et al . 2006). In agreement with these studies, our results also showed that a significant difference in response of P N and WUE to light between transgenic cotton and non-transgenic cotton did not always occur throughout the growing season which was in agreement with our work showing that the responses of P N and WUE to CO 2 were highly growth-stage-dependent (Sun et al. 2009).

Growth-stage variation in the response of P N and WUE to light could be caused either by the expression mechanisms of photosynthetic regulation genes having spatial and temporal characteristics or by temporal specific expression of Bt and Bt coupled with CpTI (Sachs et al . 1998, Kang et al . 2005). Transgenic cotton had imperfections such as an imbalance between source and sink (Tian and Yang 1999), less capability utilizing photosynthetic products by cotton bolls (Zhao et al . 2002) etc . Hebbar et al . (2007) reported that premature senescence could impact on growth and physiological processes of transgenic Bt cotton. We speculated that disorder in nitrogen metabolism (Sassenrath-Cole et al . 1996) and an imbalance of source and sink (Fitt et al . 1994, Wright 2004) led to transgenic cotton responding to senescence in a different way, probably through a possible accelerated senescence phenomenon at the end of the growing season. The progressive loss of chloro-

PHOTOSYNTHETIC CHARACTERISTICS OF INSECT-RESISTANT TRANSGENIC COTTON IN RESPONSE TO LIGHT 407

plast membrane integrity coupled with increased leaf waxiness (Bondada and Oosterhuis 2002), breakdown of Rubisco protein (Jiang et al . 1993), decreases in levels of leaf nitrogen, soluble protein, chlorophyll, photosynthetic enzymes and RNA synthesis (Evans 1983, Wells 1988, Wullschleger and Oosterhuis 1990) may limit photo-synthetic activities of cotton leaves during senescence. On the other hand, the structural and biochemical changes of the leaf could have effects on photosynthesis though variation in the partitioning of incoming radiation into reflectance, absorption and transmittance (Kakani et al . 2004). Since all cotton varieties showed similar changes in g s and LUE app at different stages, the differences in g s and LUE app response to light between transgenic cotton and non-transgenic parental counterpart could not be explained by any stage-related trend.

Our results showed that the responses of P N and WUE to light observed in the pot experiment differed from those observed under field conditions. The reason for such discrepancies may be due, in part, to profound dif-ferent effects of microclimate on cotton between transgenic varieties and non-transgenic varieties. P N , g s and various other photosynthetic characteristics are influenced by numerous environmental and physiological factors. Although these effects are often highly species-dependent, many studies also indicated that the conditions under which a plant develops could exert a significant influence on its photosynthetic charac-teristics (Bunce 1985, Schulze 1986, Wells 1988, Wullschleger and Oosterhuis 1990). For example, environmental factors can induce changes in leaf internal structure that are associated with a decrease in photo-synthesis (Kakani et al . 2004). Variation of numerous environmental factors, such as temperature (Traore et al . 2000), CO 2 concentration (Coviella et al . 2002, Wu et al . 2007), water (Matzke et al . 1990, Traore et al . 2000), methods of fertilizer application, and cultivation management (Bruns and Abel 2003) could lead to change

either in transgene expression or in growth and physio-logical processes within transgenic crops. We speculate that culture environment could affect photosynthesis of transgenic cotton both by a direct pathway and in an indirect manner through transgene expression. However, our study cannot distinguish these effects from canopy environment and the intrinsic metabolic processes of transgenic cotton.

The introduction of transgenic crops and accompany-ing changes in management practices may have potential effects on agroecosystems (Hoffman 1990, Trevors et al . 1994). It is obvious that environmental factors must be given full consideration in the safety assessment of transgenic crops. Optimisation of environmental factors and the cultivation practices of transgenic crops are expected to allow the achievement of maximal economic benefit and ecological benefit from transgenic crop production by identifying interactions between transgenic crops, environmental factors and cultivation practices.

Photosynthesis represents the final result of the complex interaction of numerous processes, any of which may be influenced by various environmental factors either directly or indirectly. It is worth mentioning that our research merely focused on photosynthetic changes based on an individual leaf throughout the growing season. Photosynthetic ability of the crop may also be affected by the structure of the crop canopy such as leaf structure, leaf shape, leaf area, plant type etc . (Heitholt 1994, Sassenrath-Cole 1995). When analyzing responses of photosynthetic characteristics to light at the whole plant or population level, it is also necessary to take into account possible effects due to canopy structure, conse-quences of changes in the light gradient within the leaf or differential acclimation of leaf surfaces to incident light (Terashima and Saeki 1985, Stirling et al . 1993), parti-cularly for crops as morphologically complex as cotton with the indeterminate growth habit. Additional investi-gations are needed to examine these issues in more depth.

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对转基因棉花的浅析

对转基因棉花的浅析 摘要：1990年，美国利用生物技术，合成B.t杀虫基因，导入棉花获得抗虫转基因棉花，成为世界上第一个拥有转基因抗虫棉的国家。我国“抗虫棉研究”在“七五”期间开始进行。“八五”期间，在“863”计划资助下，也人工合成了CryIA(b)和CryIA(c)杀虫基因导入我国棉花主栽品种获得成功，成为继美国之后，第二个拥有自主研制抗虫棉的国家。 关键词：转基因；棉花；抗虫；种植 正文： 转基因棉花是指把其他物种中的有用基因导入棉花的基因组后，获得了该基因功能的棉花。1990年，美国利用生物技术，合成B.t杀虫基因，导入棉花获得抗虫转基因棉花，成为世界上第一个拥有转基因抗虫棉的国家。我国“抗虫棉研究”在“七五”期间开始进行。“八五”期间，在“863”计划资助下，也人工合成了CryIA(b)和CryIA(c)杀虫基因导入我国棉花主栽品种获得成功，成为继美国之后，第二个拥有自主研制抗虫棉的国家。“九五”开始，“抗虫棉”的研究又被国家“863”计划立为重大项目，进一步开展单价基因、双价基因及多价基因抗虫棉的研究，分离新的抗虫基因及抗刺吸式口器害虫（蚜虫）新基因的分离，改造及合成融合蛋白基因，对获得的新基因进行重组，构建高效植物表达载体。同时“九五”期间，“863”计划还将根据目前单价抗虫棉可能存在的棉铃虫产生抗性的问题，在生产中使用的持久性问题，环境释放安全性问题，遗传分离及稳定性问题，以及田间加代选择农艺性状以及蚜虫危害等问题作深入的研究。（中国转基因棉花产业化背景） 1、转基因棉花的现状 1.1我国的转基因棉花情况 来自中国农业科学院棉花研究所的最新统计显示，今年全国转基因抗虫棉种植面积达4000多万亩，占棉花总面积一半以上。今年全国转基因抗虫棉种植面积达4656万亩，而国产转基因抗虫棉种植面积达到70％左右。目前全国种植转基因抗虫棉的省份有11个。其中河北、山东、河南、安徽4省实现了100％的种植,据中国农业科学院棉花研究所有关负责人介绍，我国目前已经建成高效、工厂化的棉花转基因技术体系，培育成功的转基因棉花新品种已达8个，年产转

转基因抗虫棉

转基因抗虫棉的研究进展 摘要：综述了转基因抗虫棉的研究进展，包括抗虫基因的研究、载体构建技术的研究、转化技术的研究及存在的问题等，并展望了转基因抗虫棉未来发展前景。关键词：转基因抗虫棉花研究进展 引言 棉花生长周期长、虫害多，造成的损失非常严重。据统计，在转基因抗虫棉商品化之前，全球每年用于防治棉花虫害的费用高达20亿美元，约占所有农作物防虫费用的四分之一。[1]传统的化学农药防治棉铃虫不仅费用高，且已引发了棉虫的抗药性，同时化学杀虫剂的过量使用也带来了环境污染的问题，而转基因植物所产生的杀虫蛋白主要是通过抑制害虫消化等生理功能而达到抗虫的目的。与施药防治棉田害虫相比，转基因技术具有较多优势：不会在土壤和地下水中造成残留；不会被雨水冲刷流失；对非靶标生物无毒性；保护作用无盲区；减少农药及用工投入[2]等。雪花凝集素（Gulanthus nivalis agglutinin gene，GNA）是第一个转入重要作物、并对刺吸式口器害虫有抗性的基因，转GNA的水稻可降低害虫的存活率，阻止害虫的发育[3]。另外烟草阴离子过氧化物酶[4]、昆虫几丁质酶基因[5]也被用于抗虫基因工程的研究。迄今为止在棉花抗虫基因工程研究领域，最成功的例子是苏云金芽孢杆菌Bt杀虫基因的应用，其次是蛋白酶抑制剂基因。另外，凝集素、α-淀粉酶抑制剂、胆固醇氧化酶等转基因抗虫植物的研究也取得了进展，所以利用基因工程技术培育转基因抗虫棉受到了各国的高度重视。自1996年商品化种植转基因作物开始，全球转基因植物的种植面积已由1996年的170万hm2猛增到2008年的1.25亿hm2，增长了73倍，2008年全球市场价值已达75亿美元，约占全球商业种子市场的22%，其市场价值优势明显，转基因产业得到了蓬勃发展，尤其在发展中国家。印度Bt棉2002年引入，连年种植面积快速增加，至2008年达760万hm2，产量翻番，曾经是全球棉花产量很低的国家，现已成为棉花出口国。转基因棉回报率高，生产成本低，市场需求大，到2005年我国通过国家审定的转基因抗虫棉品种已增至26个[6]，到2007年我国转基因抗虫棉种植面积已达380万hm2，占全国棉花种植面积的69%。农业生物技术应用国际服务组织（ISAAA）发布的报告显示，2007年转基因作物种植面积增加了12%，达到了1.143亿hm2，成为过去五年来种植面积增加第二快的一年。[7] 2008年至2010年，我国新型转基因抗虫棉培育和产业化全面推进，新培育36个抗虫棉品种，累计推广1.67亿亩，实现效益160亿元，国产抗虫棉市场份额达到93%，有效控制了棉铃虫危害，彻底打破了国外抗虫棉的垄断地位。这是我国转基因生物新品种培育重大专项取得的成就之一。我国转基因技术研究与应用取得了显著成效：获得一批具有重要应用价值和自主知识产权的基因，培育一批抗病虫、抗逆、优质、高产、高效的重大转基因生物新品种，为我国农业可持续发展提供强有力的科技支撑。 然而，随着转基因抗虫棉在世界范围的发展，也不断涌现出一些问题，如棉花质量问题，抗虫性持久问题，对生态环境安全问题等，本文综述了转基因抗虫棉的研究进展，包括抗虫基因的研究、载体构建技术的研究、转化技术的研究及存在的问题等，并展望了转基因抗虫棉未来发展前景。 1.几种抗虫基因的介绍 1.1 Bt基因 Bt基因是苏云金芽孢杆菌（Bacillus thuringiensis）的简称，它是一种革兰氏阳性菌，在地球上分布十分广泛。Bt基因是抗虫棉中研究最多、进展最快、应用最广泛的一类基因，它对鳞翅目昆虫有专一杀伤作用。大部分Bt杀虫蛋白具有杀虫专

转基因棉花的应用研究

转基因棉花的应用研究 1.引言 自1990年美国合成苏云金芽胞杆菌基因（BT基因）并导入棉花获得世界上第一株抗虫转基因棉花以来，世界各国纷纷展开对转基因农作物的研究，发达国家把发展转基因技术作为抢占未来科技制高点和增强农业国际竞争力的战略重点，发展中国家也积极跟进。研究的主要方向分别是抗虫、耐除草剂、抗病、纤维改良、抗旱和耐盐碱[1]。 上个世纪90年代，在中国大部分种植棉花的地区持续性发生棉铃虫害，给棉花生产带来了巨大的威胁，大大增加了棉花种植成本，给种植棉花的农户造成了一定的经济损失。“八五”期间，中国人工合成的Cry1Ab和Cry1Ac杀虫基因导入棉花中获得成功，成为了世界上继美国之后第二个拥有自主研制抗虫棉的国家[2]。培育更加具备优良性状的转基因棉花品种，应用于棉花生产，解决棉花 病虫害给棉花生产带来的巨大损失，减少化学农药的使用量，保护环境和生态平衡，成为了当代转基因技术研究工作者的目标。 2.转基因棉花的技术来源与应用实践 2.1 转基因技术介绍 随着生物基因技术的出现，在技术工具理性的驱动下，人类获得了打破物种间固有边界并根据人类自身的意愿重新改造生物的能力。转基因技术的原理是将人工分离或修饰过的优质基因，导入到生物体基因组中，从而达到改造生物的目的。其理论基础来源于进化论衍生来的分子生物学[3]。基因片段的来源可以是 提取特定生物体基因组中所需要的目的基因，也可以是人工合成指定序列的DNA 片段。 转基因技术的应用可以使重组生物增加人们所期望的新性状，培育出新品种。转基因作物的大规模商业化种植始于1996年，主要的转基因作物有棉花、大豆、玉米和油菜[4]。转基因技术的遗传转化方法按是否需要通过组织培养分成两大类，一类需要通过组织培养再生植株，常用的方法有农杆菌介导转化法、基因枪法；另一类方法不需要通过组织培养，比较成熟的主要有花粉管通道法[5]。 2.2转基因棉花介绍 棉花，属于锦葵科棉属，是世界上最主要的经济作物之一。全世界植棉国家和地区有96个，其中产量较高的国家有中国、美国、印度等[6]。中国是世界产棉大国，年植棉面积470万公顷，棉花产量占世界总产量的1/4[7]。病虫害对棉花的种植有很大的影响，制约了棉花的发展。与此同时，化学农药的大量使用导致了一些棉花害虫抗药性的产生，严重威胁了棉花的生产，并且使环境污染日益恶化。 随着转基因技术的发展，利用植物基因工程和遗传育种技术手段培育的转基因抗虫、耐除草剂棉花，为棉花害虫和草害的控制提供了新的手段。转基因棉花受到了棉农的喜爱，得到了进一步的推广。转基因棉花，就是将人们预想的可以得到表达的基因（比如抗病虫害的苏云金芽胞杆菌基因）通过遗传转化方法进行

转基因抗虫棉的研究历程与展望

中国转基因抗虫棉的研究历程与展望——基因工程研究进展作业

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棉花转基因技术研究进展 摘要：近些年来，转基因技术被广泛应用于遗传育种中，尤其是在棉花遗传育种和基因改良方面取得了重大的突破，本文主要通过查阅参考文献，综合阐述了三大转基因技术手段及其在棉花抗虫、抗病、抗除草剂以及品质改良等方面的应用与最新进展，并对棉花转基因研究进展中存在的主要问题进行分析。 关键词：棉花；转基因；育种；外源基因；进展 转基因技术又称外源基因导入技术, 它是通过采用农杆菌介导技术、花粉管通道技术与基因枪导入技术, 将外源基因导入受体作物的细胞，并得到整合与表达的一种现代高新育种技术。在国家“863”项目的支持下，我国自20世纪80年代末开始棉花转基因育种技术研究与开发应用,该项技术在我国发展很快，也取得了一些重大的成就与技术突破。在转基因技术的正确指引下，目前棉花转基因育种中已获得了大量转基因抗病、抗虫、抗除草剂等方面的优良抗性株系,并选育出了多个转基因抗病虫品种,这些品种在增强抗病虫性的同时，基本上延续了受体亲本原有的高产、优质的良好性状，但所选育的转基因棉花品种仍不同程度地存在种子活力低、前期生长发育偏慢、中期易发生茎枯病、后期易脱肥早衰、抗性范围窄且随生育进程强度下降等缺陷。 一、转基因棉花研究的概况 自从1983年，人类首次获得转基因烟草、马铃薯以来，植物转基因技术在基础研究和应用开发中获得了显著进展，成功培育一批具有抗虫、抗病、耐除草剂和高产优质等外源优异性状的农作物新品种，对农业的生产方式和经济效益产生了深刻影响。棉花是利用转基因技术进行遗传改良最为成功的作物之一，仅我国自主研制的CryA+CpTI双价抗虫等基因就已被转育到41个棉花品种中。棉花作为主要的经济作物之一，对一个国家国民经济发展起到了举足轻重的作用，也曾一度被列入我国战略储备物资行列。近年来，随着基因工程技术的不断发展，利用生物技术来创新棉花种质资源和培育新品种是一条非常有效的途径，极大地推动了棉花遗传育种的发展随着国际转基因技术的不断发展和转基因生物安全性方面的相关法律法规的不断完善，转基因作物的大面积推广和种植势不可挡。美国转基因抗虫棉大田种植已超过其棉田总面积的70%，澳大利亚和中国超过30%，全球转基因棉花种植面积达到680万公顷，占世界棉花种植面积的20%。 棉花为我国主要农作物之一，其基因工程方面的研究进展迅速，在抗虫、抗病、抗除草剂、棉纤维品质改良和生态效应方面均取得了一系列重要研究成果。1992 年中国农科院

棉花转基因

基金项目：863子项目“特殊生境植物资源的开发利用技术”（No ：2007AA021401）转基因专项“转新型基因的棉花种质资源材料创造”（No ：2008ZX08005-004）。 第一作者简介：张煜星，男，1967年出生，副教授，博士生，从事植物基因工程研究。通信地址：571101海口市城西学院路，中国热带农业科学院热带生物技术研究所国家重点实验室周鹏转张煜星，Tel ：015109875070，E-mail ：zyx2027193@https://www.sodocs.net/doc/0916210153.html, 。 通讯作者：男，1963年出生，研究员，博士生导师，从事植物基因工程研究。E-mail ：Zhp6301@https://www.sodocs.net/doc/0916210153.html, 。 收稿日期：2009-04-16，修回日期：2009-05-07。 棉花accD 基因植物表达载体的 构建与遗传转化的研究 张煜星1,2,3，崔燕3，祝建波3，周鹏2 （1海南大学农学院，海南儋州571737； 2中国热带农业科学院热带生物技术研究所国家重点实验室，海口571101； 3石河子大学生命科学学院农业生物技术重点实验室，新疆石河子832003） 摘要：乙酰辅酶A 羧化酶(ACCase)催化脂肪酸合成的第一步，是脂肪酸合成的限速酶。采用PCR 方法分别从陆地棉和拟南芥基因组中扩增出ACCase 羧基转移酶β-CT 亚基编码基因accD ，ACCase 羧基转移酶α-CT 亚基编码基因CAC3的定位于叶绿体的转运肽序列。将CAC3基因转运肽序列与accD 基因进行体外重组，构建融合植物表达载体pBI-CAC3tp-accD 。重组质粒通过冻融法转化根癌农杆菌GV3101。渗透法转化拟南芥，收种子，在含卡那霉素的MS 培养基中发芽筛选。用叶盘法转化烟草，经不定芽诱导、生根培养，获转基因烟草植株。T1代转基因拟南芥和转基因烟草植株经卡那霉素检测、PCR 、RT-PCR 检测后，初步表明目的基因已在植株中转化成功，并可以正常转录。 关键词：陆地棉；拟南芥；accD 基因 中图分类号：S336文献标识码：A 论文编号：2009-0791 Construction of Plant Expression Vector on accD Gene fom Gossypuum hirsutum and Its Genetic Transformation Zhang Yuxing 1,2,3,Cui Yan 3,Zhu Jianbo 3,Zhou Peng 2(1College of Agronomy,Hainan University ,Danzhou Hainan 571737; 2National Key Biotechnology Laboratory for Tropical Crops, Institute of Bioscience and Biotechnology,CATAS,Haikou,571101; 3Laboratory of Agricultural Biotechnology ,College of Life Science,Shihezi Universit ,Shihezi Xinjiang 832003) Abstract:The acetyl-CoA carboxylase (ACCase)is the rate-limiting enzyme of fatty acids synthesis.The accD Gene and transit peptide of CAC3Gene was amplified from Gossypuum hirsutum Genome and Arabidopsis thaliana Geneome.Plant Expression Vector of Fusion Transit Peptide of CAC3Gene and accD Gene was constructed.The Vector of pBI-CAC3tp -accD were transferred into Agrobacterium tumefaciens https://www.sodocs.net/doc/0916210153.html,ing infiltration and leaf discs method,the gene were transferred into Arabidopsis thaliana and tobacco cell.The seeds of Arabidopsis thaliana were selected on solid medium containing Kanamycin.Transferred leafs of tobacco were selected on solid medium containing Kanamycin.Transgenic Arabidopsis thaliana (T1)and tobacco plants were found containing purpose gene by PCR.After RT-PCR,it shows that the CAC3tp -accD gene could transcript normally.Key words:Gossypuum hirsutum,rabidopsis thaliana,accD Gene 中国农学通报2009,25(18):36-40 Chinese Agricultural Science Bulletin

转基因抗虫棉实验

转基因抗虫棉实验 一转基因植株的获得 1.选取克隆良好的抗虫棉基因，并构建转基因载体 抗虫棉的基因来自细菌，属于原核生物的基因，直接分离就行 用来构建载体的是一般是一些病毒的环形基因，用限制性核酸内切酶分别对该环形基因和目的基因进行切割，将切好的目的基因（抗虫棉基因）和环形基因通过DNA连接酶进行连接构成一个完整的基因，这个基因就是带有抗虫棉基因的重组基因 2.将目的基因转入植物细胞 主要方法有：1.鸟枪法 2.根癌农杆菌转化 3.慢病毒转染 农杆菌介导转化过程 农杆菌介导转化过程主要分为两步： 首先：农杆菌将T-DNA以单链的形式从Ti质粒上切下，然后与一系列Vir蛋白接个，这些过程都发生在农杆菌细胞内； 然后：与T-DNA相连的vir E2蛋白上有核定位序列，在它的作用下，T-DNA被转入宿主细胞，然后整合宿主基因组，整合的方式主要有双链打开修复和单链缺口修复两种方式。 3.再生植株培养（详细请查阅相关课本） 4.再生植株移苗至盆栽 二转基因植株分子检测 1.取再生植株叶片，进行基因组DNA提取 1 设备：移液器，冷冻高速离心机，台式高速离心机，水浴锅，陶瓷研钵，50ml离心管（有盖）及5ml和1.5ml离心管，弯成钩状的小玻棒。 2试剂 1、提取缓冲液Ⅰ：100mmol/L Tris·Cl, pH8.0, 20mmol/L EDTA, 500mmol/L NaCl, 1.5% SDS。

2、提取缓冲液Ⅱ：18.6g葡萄糖，6.9g二乙基二硫代碳酸钠，6.0gPVP，240ul巯基乙醇，加水至300ml。 3、80:4:16/氯仿:戊醇:乙醇 4、RnaseA母液：配方见第一章。 5、其它试剂：液氮、异丙醇、TE缓冲液，无水乙醇、70%乙醇、3mol/L NaAc。 3操作步骤： （一）水稻幼苗或其它禾木科植物基因组DNA提取 1. 在50ml离心管中加入20ml提取缓冲液Ⅰ, 60℃水浴预热。 2. 再生植株叶片5-10g, 剪碎, 在研钵中加液氮磨成粉状后立即倒入预热的离心管中, 剧烈摇动混匀, 60℃水浴保温30-60分钟(时间长,DNA产量高), 不时摇动。 3. 加入20ml氯仿/戊醇/乙醇溶液, 颠倒混匀(需带手套, 防止损伤皮肤)，室温下静置 5-10分钟, 使水相和有机相分层(必要时可重新混匀)。 4. 室温下5000rpm离心5分钟。 5. 仔细移取上清液至另一50ml离心管,加入1倍体积异丙醇,混匀,室温下放置片刻即出现絮状DNA沉淀。 6. 在1.5ml eppendorf中加入1ml TE。用钩状玻璃棒捞出DNA絮团,在干净吸水纸上吸干,转入含TE的离心管中,DNA很快溶解于TE。 7. 如DNA不形成絮状沉淀,则可用5000rpm离心5分钟, 再将沉淀移入TE管中。这样收集的沉淀,往往难溶解于TE,可在60℃水浴放置15分钟以上，以帮助溶解。 8. 将DNA溶液3000rpm离心5分钟, 上清液倒入干净的5ml离心管。 9. 加入5μl RNaseA(10μg/μl), 37℃10分钟, 除去RNA(RNA对DNA的操作、分析一般无影响，可省略该步骤）。 10. 加入1/10体积的3mol/L NaAc及2×体积的冰乙醇,混匀,-20℃放置20分钟左右,DNA 形成絮状沉淀。 11. 用玻棒捞出DNA沉淀,70%乙醇漂洗,再在干净吸水纸上吸干。 12. 将DNA重溶解于1ml TE, -20贮存。

转基因棉花在生产应用上的风险与安全控制

转基因棉花在生产应用上的风险与安全控制 植物转基因技术的诞生，对生物工程技术和世界农业都产生了巨大影响。棉花是纤维植物，由于遗传转化的限制，棉花的转基因研究起步较晚，但近几年进展迅速。一方面，随着转基因技术的发展，抗虫、抗病、抗除草剂基因等先后成功导入棉花载体中并应用于大田生产，使得转基因抗虫棉成为我国唯一大面积种植并实现产业化的转基因农作物。对缓解棉铃虫给棉花生产造成的为害、减少化学农药的使用量和保护生态环境起到了重要的作用；另一方面，随着转基因棉花的大面积应用，其安全性问题逐渐引起了人们的重视，转基因棉花的田间种植是否会引起外源基因向其他物种渗透，是否会对生态环境造成危害，这都是转基因植物进入商业化生产前都必须明确的问题¨1。但目前人们还很难准确预测外源基因导入一个新的遗传背景会产生什么样的后果心1。因此，必须采取措施预防转基因棉花在生产应用中可能存在的安全风险，作者以目前应用面积最大的转Bt基因抗虫棉为基础，进行转外源基因棉花在生产上应用对生态环境安全的影响分析，并提出转Bt基因抗虫棉在生产应用上的风险预防和安全控制措施。 1 转基因棉花在生产应用上的风险

1.1抗除草剂棉花有成为杂草的可能 杂草生长迅速并且具有强大生存竞争力，能够广泛传播，阻碍农作物的生长。所以，杂草常常给农业生产造成巨大损失。虽然转基因棉花在抗病虫性、抗逆性和生活力等方面比非转基因棉花强，但由于棉花不具有杂草特性，不会入侵其他植物栖息地，破坏自然种群平衡，一般情况下，转基因棉花不会出现杂草化问题。但近年来棉田除草普遍使用除草剂对棉花造成伤害，为了减少除草剂对棉花的伤害而开展的抗除草剂棉花选育。有使其成为杂草的可能，如果抗除草基因从棉花漂移到杂草上，也就有可能出现抗（耐）除草剂的“超级杂草”。1.2基因漂移威胁棉花近缘物种基因漂移是指基因通过花粉授精杂交等途径在种群之间扩散的过程。棉花是常异花授粉作物，转基因棉花的外源基因花粉可以通过风力、昆虫等向近缘非转基因植物转移，不仅影响近缘物种的遗传纯度，使近缘物种有获得选择优势的潜在可能性，如果近缘物种获得抗病、抗虫或抗除草剂基因。也可能使其成为另一种“超级杂草”。这样会促使大量化学农药的应用，造成严重的环境危害。另外，随着转基因棉花的大面积推广应用，还可能向其他物种渗透，若大量外源基因漂移进入野生植物基因库并扩散开来，可能会影响基因库的遗传结 构，给生物多样性造成危害心1。

转基因技术及其在棉花育种中的应用

转基因技术在棉花育种中的应用 杨金惠 812031001 作物领域 2012级 摘要：棉花是一种重要的经济作物，在我国广泛种植。培育转基因棉花被看作是解决产量和生态环境问题最根本和最有效的方式。本文介绍了转基因棉花主要的研究方法，包括转化方法以及转入的基因等，并对转基因棉花的发展趋势作了相关探索。此外，本文总结了转基因技术在棉花遗传改良中的应用，包括棉花抗病、抗虫、抗除草剂、抗逆以及品质改良等方面的最新进展，并对棉花转基因研究中存在的主要问题和今后的研究与应用前景进行分析和展望。 关键词：转基因；棉花；育种 1973 年美国科学家科恩等人第一次将两种不同的DNA 分子进行体外重组, 并且在大肠杆菌中表达以来, 基因工程技术发展飞速, 该技术正在极大地改变着地球生物固有的进化进程。据不完全统计, 目前全球已有60 多种转基因园艺植物和大田作物相继问世, 其中转基因工程技术在棉花品种改良中的应用, 成效卓著。 自从1983年人类首次获得转基因烟草、马铃薯以来，植物重组DNA技术在基础研究和应用开发中获得了显著进展，培育成功一批具有抗虫、抗病、耐除草剂和高产优质等外源优异性状的农作物新品种，对农业的生产方式和经济效益产生了深刻影响。棉花是利用转基因技术进行遗传改良最为成功的作物之一，仅我国自主研制的,CryA+CPTI双价抗虫等基因就已被转育到41个棉花品种中。美国转基因抗虫棉大田种植已超过其棉田总面积的70%，澳大利亚和中国超过30%，全球转基因棉花种植面积达到680万公顷，占世界棉花种植面积的20%。 1.转基因技术 棉花转基因技术是指将外源DNA通过物理、化学或生物学方法导入棉花细胞并得到整合和表达的过程。在棉花遗传转化体系中，主要有农杆菌介导、花粉管通道和基因枪3种转化方法。本研究拟对. 种方法的主要技术特点及研究和应用动态进行综述，旨为棉花分子育种提供参考。 1.1．农杆菌介导法 1.1.1农杆菌转化技术的理论基础 与棉花遗传转化有关的根癌农杆菌是一种土壤习居菌，在自然状态下能感染棉花等大多数双子叶植物营养器官的伤口，导致冠瘿瘤的发生。根癌农杆菌含有一种Ti质粒，侵染时通过棉花器官的伤口进入寄主组织，但其本身不进入寄主植物细胞，只把Ti质粒的T-DNA片断导入棉花的基因组中并得以表达，且外源DNA 表达通常表现出典型的孟德尔遗传规律。由于Ti质粒本身能插入大到50kb的外源DNA，因此利用此转化载体，将Ti质粒上致瘤基因切除，代之以有益的外源DNA 序列，并插入由真核型启动子和细菌抗生素抗性选择标记基因或报告基因组成的嵌合基因，将改造后的农杆菌侵染棉花器官或细胞，在加有相应选择因子的培养基上选择转化再生植株，进而可得到转基因植株。 1.2.花粉管通道法 1.2.1花粉管通道转化技术的理论基础 从整体上说，远缘亲本间的染色体结构是不亲和的。但从进化的角度来看，任何生物DNA均由4种核苷酸组成，这样就可能在顺序上出现不同程度的相同排

转基因棉花

转基因抗虫棉的发展以及存在的问题 张文亮26 摘要：棉花是重要的农作物以及经济产物，自上个世纪90年代以来， 由于棉铃虫在我国大部分棉区持续性大发生或爆发，给棉花生产带来 了巨大的威胁，因此开始进行抗虫棉的研究。本文叙述了抗虫棉的发 展过程以及抗虫棉的特点，从而对转基因抗虫棉有了更深刻的了解， 不仅仅停留在其优点，也发现了它的潜在危害，毕竟所有事物都是有 两面性的。 关键词：转基因棉花、抗虫棉、抗虫性、Bt蛋白、 Abstract：Cotton is one of the important crops and economic product, since the 90s of last century, because of the cotton bollworm in major cotton growing areas of China continuing occurrence or outbreak, cotton production has brought great threat, so start study on insect resistant cotton. This paper describes the Bt cotton in the development process and characteristics of the insect resistant cotton, thus of insect resistant transgenic cotton have more profound understanding, not only stay in the utility model has the advantages of, also found the potential harm, after all, all things are two sides.