转基因棉花外源基因的遗传

选用不同来源的转Bt基因抗虫棉中棉所30号和新棉33B、转tfdA基因抗除草剂棉花材料TFD,通过与不同主栽推广品种正反交,研究了外源Bt基因和tfdA基因在棉株体内的遗传和分离行为。结果表明,由于外源基因提供给转基因棉花的抗虫和抗除草剂性状均是由一对于基因控制的显性性状,且该性状不受细胞质效应的影响,符合孟德尔遗传规律。     

转基因棉花的遗传研究

选用不同来源的转Ｂｔ基因抗虫棉中棉所３０号和新棉３３Ｂ、转ｔｆｄＡ基因抗除草剂棉花材料ＴＦＤ,通过与不同主栽推广品种正反交和回交研究外源Ｂｔ基因和ｔｆｄＡ基因在棉花体内的遗传和分离行为。结果表明,由外源基因提供给转基因的抗虫和抗除草剂性状均是由一对基因控制的显性性状,且该性状不受细胞质效应的影响,符合孟德尔遗传规律。     

Measuring gene flow in the cultivation of transgenic cotton (Gossypium hirsutum L.)

Transgenic Bt cotton NewCott 33B and transgenic tfd A cotton TFD were chosen to evaluate pollen dispersal frequency and distance of transgenic cotton (Gossypium hirsutum L.) in the Huanghe Valley Cotton-producing Zone, China. The objective was to evaluate the efficacy of biosafety procedures used to reduce pollen movement. A field test plot of transgenic cotton (6×6 m) was planted in the middle of a nontransgenic field measuring 210×210 m. The results indicated that the pollen of Bt cotton or tfd A cotton could be dispersed into the environment. Out-crossing was highest within the central test plot where progeny from nontransgenic plants, immediately adjacent to transgenic plants, had resistant plant progeny at frequencies up to 10.48%. Dispersal frequency decreased significantly and exponentially as dispersal distance increased. The flow frequency and distance of tfd A and Bt genes were similar, but the pollen-mediated gene flow of tfd A cotton was higher and further to the transgenic block than that of Bt cotton (χ² = 11.712, 1 degree of freedom, p<0.001). For the tfd A gene, out-crossing ranged from 10.13% at 1 m to 0.04% at 50 m from the transgenic plants. For the Bt gene, out-crossing ranged from 8.16% at 1 m to 0.08% at 20 m from the transgenic plants. These data were fit to a power curve model: y=10.1321x ^−1.4133 with a correlation coefficient of 0.999, and y=8.0031x ^−1.483 with a correlation coefficient of 0.998, respectively. In this experiment, the farthest distance of pollen dispersal from transgenic cotton was 50 m. These results indicate that a 60-m buffer zone would serve to limit dispersal of transgenic pollen from small-scale field tests.     

转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征。合成了Cry1Ac活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K。构建了含Bt29K基因及慈菇蛋白酶抑制剂B（API-B）基因表达框的双抗虫基因植物表达载体。通过根癌土壤杆菌（Agrobacterium trmefaciens(Smith et TOwnsend)ConnLBA4404)介导转化了棉花（Gossypium hirsu-tunL.)的两个生产品种（系）。根据抗棉铃虫（Heliothis armigera)试验及农艺性状的观察调查结果。经6代筛选,获得了抗棉铃虫90.0%_99.7%且农艺性状优良的9个双价抗虫棉纯合品系。分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个,活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%。对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的。     

外源基因导入对棉花烟粉虱种群的影响

以转基因棉花和对应的常规棉花亲本为材料,探讨外源基因导入对棉花烟粉虱Bemisia tabaci(Gennadius)种群的影响。结果表明,在转基因棉花GK12、33B、SGK上,烟粉虱从卵发育到成虫羽化的历期分别为15.56、15.35和15.25 d,较对应的常规棉亲本SM3(19.38 d)、33(20.81 d)、SY321(18.76 d)分别短24.55%、26.23%、18.71%;GK12和33B棉花上烟粉虱的存活率(69.16%)分别较对应的常规棉亲本SM3(54.76%)和33(64.91%)高26.29%和12.81%,而SGK(63.21%)上烟粉虱的存活率与对应的常规棉亲本SY321(62.61%)之间差异不显著;烟粉虱在GK12(84.00)和33B(77.25)上的产卵量分别较SM3(62.25)、33(70.00)高34.93%和10.35%,但SGK和SY321之间差异不显著;在转基因棉花GK12、33B、SGK上,烟粉虱的雌雄性比分别比对应的常规棉亲本高26.71%、46.23%和19.17%。结果表明,外源基因导入后,有助于烟粉虱的发育,提高了烟粉虱的产卵量和雌雄性比,从而促进了烟粉虱种群的上升。     

转新型双抗虫基因棉花的遗传分析

首次用含合成的BtCrylAc活性杀虫蛋白嵌合基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体,通过土壤根癌杆菌介导转化棉花品种冀合321,获得一批抗虫的转化再生棉花植株。利用叶片涂抹卡那霉素、叶片离体养虫和PCR扩增等检测方法对6个不同转双抗虫基因株系的抗虫性进行遗传分析。结果显示,转化株系自交的T1抗虫性状遗传较为复杂;农杆菌介导获得的转基因抗虫棉在早期世代不易选到纯合系,但是随着对抗虫性状进行单向选择,到T4和T5就能获得抗虫纯合系。利用抗虫性稳定的转化后代材料和转化受体进行田间杂交,发现F2抗虫性分离完全符合一对或两对显性基因的分离规律,并证明了DR248和DR193两个材料为外源基因双拷贝插入。转化株系的Southern杂交也证明了上述结果。     

转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征,合成了CrylAc活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K.构建了含Bt29K基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体.通过根癌土壤杆菌(Agrobacteriumtumefaciens(Smith et Townsend)Conn LBA4404)介导转化了棉花(Gossypium hirsu-tun L.)的两个生产品种(系).根据抗棉铃虫(Heliothis armigera)试验及农艺性状的观察调查结果,经6代筛选,获得了抗棉铃虫90.0%～99.7%且农艺性状优良的9个双价抗虫棉纯合品系.分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个.活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%.对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的.     

转Bt基因棉花及其受体品种主要挥发性物质的测定

应用顶空进样气质联机系统（Headspace-GC-MS)测定了转Bt基因棉花及其对照亲本主要挥发性物质。结果表明棉花营养器官和繁殖器官的主要挥发性物质的差异,棉花植株现蕾期前,叶片中挥发性物质以α-蒎烯为主,而现蕾后,蕾、花和铃中主要以β-月桂烯为主。研究初步发现,外源Bt杀虫蛋白表达对棉花自身主要挥发性物质的合成不会造成不利影响。     

Transformation and inheritance of Bt genes inGossypium hirsutum

Transgenic plants offer many unique opportunities for managing pest populations. However, the inheritance, integration, and expression of multiple transgenes are prerequisite for maintaining sustainable resistance against insects in crops. We took a gene-pyramiding approach to produce Bt cotton expressing two Bt genes,cry1Ac andcry2A. Using sonication-assistedAgrobacterium-mediated transformation (SAAT), we achieved an efficiency of 6.26%. Putative transgenic plants were confirmed via PCR, Southern hybridization, and western-blotting. Those showing mortality of 75 to 100% for the second instar ofHeliothis armigera (compared with 0% for the control) were considered Bt-positive. Transgenes were segregated according to a 3:1 Mendelian inheritance pattern in the T1 generation forHeliothis resistance. In our insect bioassay, the control plants showed >95% leaf damage, and insects reached the 4^th instar stage of larval growth. In contrast, leaf damage on transgenic plants was limited to only a few bites, and insect mortality was 75 to 100%. ELISA confirmed transgene expression, and Bt protein was detected in leaf tissue. This performance was consistent with that of the parent transgenics. PCR and Southern blots verified integration of thecry1Ac andcry2A genes into the progeny. Therefore, this strategy provides a pathway toward cotton improvement and the development of durable resistance against insect damage.     

棉铃虫对转Bt基因棉的抗性筛选及遗传方式的研究

通过室内筛选获得了棉铃虫Helicoverpa armigera (Hübner)对转Bt基因棉的抗性种群,并在此基础上进行了抗性遗传方式的研究。试验结果表明：经16代筛选,棉铃虫对转Bt基因棉的抗性倍数上升到43.3倍。筛选过程中,棉铃虫7日龄幼虫的体重和成虫羽化率变化明显,被选为确定筛选剂量的标准。敏感与抗性棉铃虫杂交,正交、反交的显性度都小于0、杂交过程中雌雄性比基本接近1∶1、假设抗性基因为单基因时的χ²值较低,因此,初步认为棉铃虫对转Bt基因棉的抗性是常染色体单基因控制的不完全隐性遗传。     

Effect of pyramiding Bt and CpTI genes on resistance of cotton to Helicoverpa armigera (Lepidoptera: Noctuidae) under laboratory and field conditions

Transgenic cotton (Cossypium hirsutum L.) varieties, adapted to China, have been bred that express two genes for resistance to insects, the CrylAc gene from Bacillus thuringiensis (Berliner) (Bt), and a trypsin inhibitor gene from cowpea (CpTI). Effectiveness of the double gene modification in conferring resistance to cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), was studied in laboratory and field experiments. In each experiment, performance of Bt+CpTI cotton was compared with Bt cotton and to a conventional nontransgenic variety. Larval survival was lower on both types of transgenic variety, compared with the conventional cotton. Survival of first-, second-, and third-stage larvae was lower on Bt+CpTI cotton than on Bt cotton. Plant structures differed in level of resistance, and these differences were similar on Bt and Bt + CpTI cotton. Likewise, seasonal trends in level of resistance in different plant structures were similar in Bt and Bt+CpTI cotton. Both types of transgenic cotton interfered with development of sixth-stage larvae to adults, and no offspring was produced by H. armigera that fed on Bt or Bt+CpTI cotton from the sixth stage onward. First-, second-, and third-stage larvae spent significantly less time feeding on transgenic cotton than on conventional cotton, and the reduction in feeding time was significantly greater on Bt+CpTI cotton than on Bt cotton. Food conversion efficiency was lower on transgenic varieties than on conventional cotton, but there was no significant difference between Bt and Bt+CpTI cotton. In 3-yr field experimentation, bollworm densities were greatly suppressed on transgenic as compared with conventional cotton, but no significant differences between Bt and Bt+CpTI cotton were found. Overall, the results from laboratory work indicate that introduction of the CpTI gene in Bt cotton raises some components of resistance in cotton against H. armigera, but enhanced control of H. armigera under field conditions, due to expression of the CpTI gene, was not demonstrated.     

转基因抗虫棉Bt基因不同剂量的聚合与抗虫性表现

通过有性杂交手段培育出聚有不同数目Bt基因的植株,在不同生育期进行抗虫性测定和Bt毒蛋白表达的ELISA检测,旨在揭示聚合不同数目Bt基因的植株抗虫性的互作表达机理。聚合有1－4个Bt基因的植株在整个生育期的抗虫性、毒蛋白表达特性和单价抗虫棉时空表达一致,生育前期抗虫性好、毒蛋白表达量高;生育中、后期抗虫性有所下降,毒蛋白表达量降低。聚合有4个Bt基因的纯合材料并未因Bt基因的增加而起到抗性增强的效果,相反还因同源抑制而有所降低。不同来源的Bt基因处于杂合状态时其抗虫性和Bt毒蛋白量均得到充分表达。     

Dominant Gene cplsr1 Corresponding to Premature Leaf Senescence Resistance in Cotton (Gossypium hirsutum L.)

Cotton (Gossypium hirsutum L.) premature leaf senescence-resistant inbred XLZ33 and senescencesusceptible inbred lines XLZ13 were selected and crossed to produce F1,F1-reciprocal,F2 and BC1 generations...     

Expression and inheritance pattern of two foreign genes in petunia

Transgenic petunia (Petunia hybrida Vilm.) plants were obtained from Agrobacterium-mediated shoot apex transformation. Studies at the phenotypic as well as molecular level established both the presence of the NPT II (neomycin phosphotransferase II) and GUS (-glucuronidase) genes and their level of activity. Twenty-nine primary transformed plants showed varying patterns of phenotype expression of both genes. NPT II and GUS expression in 7 primary plants over a 4-month interval showed varying levels of gene expression within and among individual plants. All primary transgenic plants were self-pollinated and backcrossed to establish the inheritance patterns of both genes. Mendelian and non-Mendelian inheritance patterns for both genes were observed. Analysis of the progeny showed poor transmission of the foreign genes through the pollen especially when two or more bands were present in the Southern hybridization. Most plants whose progeny segregated in Mendelian ratios for either the NPT II or GUS gene had just one copy of the gene. In this study where both foreign genes were examined in both self and test crosses, no transgenic plant showed Mendelian patterns of inheritance for both foreign traits.Department of Plant Pathology and Microbiology     

Transgenic cotton expressing synthesized scorpion insect toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae

A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44–98%. The AaHIT gene could used therefore an alternative to Bt toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.     

转基因抗虫棉Bt基因插入区碱基组成分析

利用TAIL－PCR的方法克隆不同来源的转基因抗虫棉中外源基因插入区的侧翼序列并对其进行序列和结构分析,结果表明,同一个较基因的单构自交得到的不同株系中外源基因插入区的两侧DNA序列完全相同,不同的转基因抗虫棉虫的外源基因插入位置各不相同,不同来源的转基因品种外源基因插入的上游侧翼片段含有一段残留质粒片段,外源基因插入的下游侧翼片段为富含AT碱基结构,其中泗棉3号转基因抗虫品系中下游侧翼片段的AT碱基高达92％,Southern杂交结果显示这些侧翼序列为高AT含量的多拷贝序列,序列中没有发现拓扑异构酶的结合位点。     

Implications of transgenic, insecticidal plants for insect and plant biodiversity

Genetically modified plants are widely grown predominantly in North America and to a lesser extent in Australia, Argentina and China but their regions of production are expected to spread soon beyond these limited areas also reaching Europe where great controversy over the application of gene technology in agriculture persists. Currently, several cultivars of eight major crop plants are commercially available including canola, corn, cotton, potato, soybean, sugar beet, tobacco and tomato, but many more plants with new and combined multiple traits are close to registration. While currently agronomic traits (herbicide resistance, insect resistance) dominate, traits conferring “quality” traits (altered oil compositions, protein and starch contents) will begin to dominate within the next years. However, economically the most promising future lies in the development and marketing of crop plants expressing pharmaceutical or “nutraceuticals” (functional foods), and plants that express a number of different genes. From this it is clear that future agricultural and, ultimately, also natural ecosystems will be challenged by the large-scale introduction of entirely novel genes and gene products in new combinations at high frequencies all of which will have unknown impacts on their associated complex of non-target organisms, i.e. all organisms that are not targeted by the insecticidal protein. In times of severe global decline of biodiversity, pro-active precaution is necessary and careful consideration of the likely expected effects of transgenic plants on biodiversity of plants and insects is mandatory.In this paper possible implications of non-target effects for insect and plant biodiversity are discussed and a case example of such non-target effects is presented. In a multiple year research project, tritrophic and bitrophic effects of transgenic corn, expressing the gene from Bacillus thuringiensis (Bt-corn) that codes for the high expression of an insecticidal toxin (Cry1Ab), on the natural enemy species, Chrysoperla carnea (the green lacewing), was investigated. In these laboratory trials, we found prey-mediated effects of transgenic Bt-corn causing significantly higher mortality of C. carnea larvae. In further laboratory trials, we confirmed that the route of exposure (fed directly or via a herbivorous prey) and the origin of the Bt (from transgenic plants or incorporated into artificial diet) strongly influenced the degree of mortality. In choice feeding trials where C. carnea could choose between Spodoptera littoralis fed transgenic Bt-corn and S. littoralis fed non-transgenic corn, larger instars showed a significant preference for S. littoralis fed non-transgenic corn while this was not the case when the choice was between Bt- and isogenic corn fed aphids. Field implications of these findings could be multifold but will be difficult to assess because they interfere in very intricate ways with complex ecosystem processes that we still know only very little about. The future challenge in pest management will be to explore how transgenic plants can be incorporated as safe and effective components of IPM systems and what gene technology can contribute to the needs of a modern sustainable agriculture that avoids or reduces adverse impacts on biodiversity? For mainly economically motivated resistance management purposes, constitutive high expression of Bt-toxins in transgenic plants is promoted seeking to kill almost 100% of all susceptible (and if possible heterozygote resistant) target pest insects. However, for pest management this is usually not necessary. Control at or below an established economic injury level is sufficient for most pests and cropping systems. It is proposed that partially or moderately resistant plants expressing quantitative rather than single gene traits and affecting the target pest sub-lethally may provide a more meaningful contribution of agricultural biotechnology to modern sustainable agriculture. Some examples of such plants produced through conventional breeding are presented. Non-target effects may be less severe allowing for better incorporation of these plants into IPM or biological control programs using multiple control strategies, thereby, also reducing selection pressure for pest resistance development.     

不同转基因抗虫棉对棉铃虫抗虫性的时空动态

利用2个对Bt抗性水平不同的棉铃虫品系,测定了转Bt和CpTI基因双价抗虫棉(SGK321)和Bt棉(GK12,33B)杀虫活性的时间和空间动态变化。结果表明,2类3种抗虫棉杀虫活性共同表现为：(1) 时间动态上均呈现前高后低的下降趋势;(2) 空间动态上表现为,在生长前期以叶的活性最高,中后期以铃和蕾的活性较高;(3) 对敏感品系的活性高于对抗性品系。不同点表现为：（1）双价棉在生长中后期(8～9月份)活性明显高于Bt棉;（2）双价棉对抗性品系的活性表现更稳定。     

双价抗虫基因叶绿体共转化植株抗虫性及其后代表型分析

利用基因枪法将含有水稻巯基蛋白酶抑制剂(Oryzacystatin,OC)基因烟草叶绿体表达载体和含有苏云金芽孢杆菌晶体毒蛋白基因(Bt cry IAc)烟草叶绿体表达载体,共转化烟草叶绿体,获得壮观霉素抗性植株。转基因植株抗棉铃虫试验表明,转双价抗虫基因植株比转单一抗虫基因植株具有更强的杀虫活性。转基因植株后代Southern检测及其遗传学分析试验证明,双价抗虫基因可以稳定地遗传给后代,且表现为叶绿体特有的母系遗传规律。 Abstract:The Bt gene and OC gene were cotransformed to tobacco chloroplast with particle bombardment method and spectinomycin resistance tobacco seedlings were obtained.Bioassays showed that the transgenic tobacco containing both genes had enhanced toxicity to the larvae of cotton bollworm (helicoverpa zea) by comparison with the plants containing only Bt or OC gene.Southern-blotting analysis and genetic analysis of progenies showed that the Bt and OC gene expressed and was inherited maternally to the progenies.     

Bt抗虫棉秸秆还田对土壤养分特征的影响

【目的】研究转基因作物秸秆或残茬还田可能对土壤养分特性造成的影响。【方法】以不同抗虫水平Bt棉花和常规棉花(泗棉3号)为研究材料,分别在经过一、二个生长周期后将秸秆机械粉碎后原位还田,40 d后测定分析土壤中Bt蛋白含量及肥力相关的养分含量变化。【结果】Bt棉秸秆还田后,所有品种棉花土壤中Bt蛋白含量与还田前无显著增加,且转Bt基因棉与非转基因棉还田对土壤Bt蛋白含量的影响并无显著差异。同时,棉秸秆还田可显著提高土壤有机质、速效磷、碱解氮、速效钾、全氮、全磷和全钾含量,提升土壤pH值;增加幅度在不同抗虫水平Bt棉花间及与非转基因常规棉花品种间皆无显著性差异。【结论】秸秆还田对土壤肥力的提升与Bt棉的抗虫水平无关。“转Bt基因”不成为Bt棉秸秆还田提高土壤肥力的限制性因素,其秸秆还田不会对土壤肥力质量产生负面影响,可使土壤养分含量增加,有效提升土壤肥力。秸秆原位还田简单、无害又提升肥力,有条件作为转Bt基因植物秸秆无害化处理的理想方式。