转Cry1Ac+Cry2Ab基因棉花对天敌生长发育的影响

 以转Cry1Ac基因棉和常规棉品种为对照,系统研究了转Cry1Ac+Cry2Ab棉花对小花蝽、龟纹瓢虫和中华草蛉生长发育的影响。结果表明,和常规棉品种相比,饲喂取食转Cry1Ac+Cry2Ab基因棉和转Cry1Ac基因棉品种叶片的棉铃虫初孵幼虫使各天敌不同龄期的生长发育历期均有所延长,前者延长的历期短,后者延长的历期长,但差异均未达显著水平。     

Cry2Ab 蛋白对棉花黄萎病菌、枯萎病菌和红腐病菌的影响

研究Cry2Ab蛋白对棉花黄萎病菌、枯萎病菌和红腐病菌生长速率及对黄萎病菌分生孢子产量、分生孢子大小和粗毒素分泌量的影响。结果发现,当Cry2Ab蛋白质量浓度为1.0 μg·mL^－1时,显著降低黄萎病菌分生孢子的大小;当其质量浓度大于5.0 μg·mL^－1时,显著抑制棉花黄萎病菌、枯萎病菌和红腐病菌在PDA培养基上生长及黄萎病菌分生孢子的大小,促进黄萎病菌产孢和粗毒素分泌。     

Cry1Ab基因转化甘蔗及转基因抗虫植株的获得

构建玉米UBI启动子驱动Bt Cry1Ab基因的高效植物表达载体pUBTB,通过农杆菌介导法导入甘蔗愈伤组织,经过除草剂PPT三次连续筛选,共获得67株抗性植株,经PCR扩增检测,得到22株阳性植株,并对其进行RT-PCR检测,证明外源基因已经成功整合到其中的15株甘蔗基因组中,且得到了有效的表达。     

基因枪介导法获得转BtCry1Ac基因抗虫玉米植株的研究

利用基因枪介导法将含有Cry1Ac基因的质粒转入玉米愈伤组织,获得转Bt基因玉米植株,对所获得的转基因植株进行PCR分析和金标免疫试纸鉴定。结果表明,部分转化植株呈阳性,说明目的基因已经整合到玉米基因组中。将转基因植株自交获得T1代种子,对T1代植株进行田间抗虫鉴定,结果表明,与对照相比转基因玉米植株的抗虫活性明显强于非转基因植株。     

转Cry1Ab基因水稻恢复系KR1128的鉴定及组合评价

螟虫是危害水稻生产的重要虫害,长江中下游稻区尤以二化螟发病较多,对水稻稳产、增产造成较大影响。研究以转Cry1Ab基因抗虫恢复系mfb-MH86为供体,强优恢复系HR1128为受体,通过田间回交转育,结合分子标记检测、农艺性状筛选得到抗虫恢复系新材料KR1128-1、KR1128-2和KR1128-3。产量性状分析结果表明,改良恢复系与非转基因对照HR1128相比,株高显著降低,但单株有效穗数有所改善,均显著高于HR1128;改良恢复系的结实率均略高于亲本HR1128,以KR1128-3的结实率最高（82.0%）。在稻米品质上,改良新株系与HR1128多数指标无显著差异,少数性状稍有改善,如KR1128-3在整精米率、垩白面积和垩白度上均显著优于HR1128。酶联免疫吸附试验显示,改良恢复系均能够稳定表达Bt蛋白,以茎秆中抗虫蛋白含量最高、穗部籽粒中最少;室内螟虫抗性鉴定结果证明,所有改良系的抗虫性均大幅提升,达到高抗水平。测配结果表明,两系杂交稻抗虫组合整体表现好于三系杂交稻组合,其中恢复系KR1128-3与Y58S、丰源A和炳1A所配组合的产量均高于其他转基因组合和HR1128与各...     

Cry1Ac和sck双价抗虫基因遗传转化甘蔗的研究

将双价抗虫基因,即修饰的苏云金芽孢杆菌毒蛋白基因(Cry1Ac)和修饰的豇豆胰蛋白酶抑制剂基因(sck)导入甘蔗优良品种ROC22,获得潮霉素抗性转化植株95株,35株能检测到外源抗虫基因,获得的13株RT-PCR双基因均呈阳性的甘蔗植株,斑点杂交检测有4株呈阳性。结果表明:外源抗虫基因已导入甘蔗基因组中。     

转Chi和Glu基因抗病棉花对棉田主要害虫和天敌的影响

 在不喷施化学药剂的条件下,对转Chi和Glu基因抗病棉田4种主要害虫蚜虫、叶蝉、粉虱、盲蝽和主要天敌瓢虫、小花蝽、蜘蛛、草蛉卵的消长进行了调查研究,并与当地常规品种冀丰106、鲁棉研28棉田进行了比较。结果表明,在生育期的大部分阶段,转Chi和Glu基因抗病棉害虫及天敌的发生数量、趋势与2个当地对照品种差异不显著,仅在个别生育期,个别害虫或天敌差异显著。这表明转Chi和Glu基因抗病棉花的短时间种植,并未对棉田生物多样性产生明显的影响。     

转Bt基因棉棉子壳培养基对平菇生长发育的影响

研究了转Bt基因棉花的棉子壳作为培养基对平菇生长发育的影响.结果表明,和常规棉对照相比,平菇在转Bt基因棉棉子壳培养基上菌丝生长缓慢,出菇期推迟3天左右,收菇期推迟2天左右,产菇量没有明显差异,但转Bt基因棉棉子壳生物转化率高于常规棉.     

转Bt基因棉花新品种冀丰106的选育

依据国家棉花育种攻关指标,以大铃、高产和抗病虫为主攻方向,通过亲本的广泛筛选,利用丰产性、抗病性突出的远缘杂交后代做母本,与结铃性强、高抗棉铃虫的新品系进行杂交,经过多年南繁北育连续选择、早代纤维品质检测、病圃抗病性鉴定以及多环境适应性鉴定等,选育出转Bt基因棉花新品种“冀丰106“.该品种2005年通过河北省审定,2006年通过天津市认定,2007年通过山西省认定,并被列入河北省棉花良种补贴项目品种,目前在棉花生产上发挥着重要作用.     

转cry1Ab基因水稻对拟水狼蛛捕食作用间接影响的评价

室内评价了拟水狼蛛(Pirata subpiraticus)对取食转[i]cry1Ab[/i]基因水稻的稻纵卷叶螟([i]Cnaphalocrocis medinalis[/i])幼虫和褐飞虱([i]Nilaparvata lugens[/i])若虫的捕食量及功能反应。结果表明,拟水狼蛛对这2种猎物的捕食功能反应均符合HollingⅡ型圆盘方程。该蜘蛛对取食转[i]cry1Ab[/i]基因水稻的稻纵卷叶螟幼虫的捕食量和处理时间低或短于取食对照的,但差异未达显著水平,而瞬时攻击率显著低于对照。该蜘蛛对以转[i]cry1Ab[/i]基因水稻为食的褐飞虱若虫的捕食量、瞬时攻击率和处理时间与取食对照的相比有一定的变化,但差异均不显著。综合考虑,认为转[i]cry1Ab[/i]基因水稻对拟水狼蛛捕食作用无明显的负作用。     

转基因抗虫抗草甘膦棉剔除杂株药剂初步研究

 为剔除转基因抗虫抗草甘膦棉中的杂株,试验采用硫酸卡那霉素4000 mg·L^-1复配41%草甘膦异丙胺盐3.3～13 mL·L^-1 4个梯度处理,以硫酸卡那霉素4000 mg·L^-1和草甘膦异丙胺盐13 mL·L^-1为2个对照,结果硫酸卡那霉素4000 mg·L^-1+草甘膦异丙胺盐3.3 mL·L^-1复配处理剔除杂株最为经济有效。同时显示该剔除液亦可用于转基因抗虫抗草甘膦棉的抗草甘膦性鉴定。     

Season-long expression of Cry1Ac and Cry2Ab proteins in Bollgard II cotton in Australia

Bollgard II cotton has been grown commercially in Australia since 2003 for control of the primary target species Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) Larvae of both species have been reported to survive at low frequencies on Bollgard II with larvae >8 mm recorded in between 7 and 18% of the area planted to Bollgard II cotton between 2005/06 and 2007/08. F₁ and F₂ tests have shown that this is not due to the presence of resistance genes in the surviving larvae. To understand if fluctuations in the expression of the Cry proteins in Bollgard II allow some larvae to survive, plant tissue samples were taken from five Bollgard II cultivars throughout the growing season within fields and from different farms within a production region between 2007 and 2010. The data indicate that the expression of both Cry proteins is similar to the known resistance-monitoring diagnostic concentrations and relatively uniform between fields within a farm and between farms within a region, with less than one-third of the tests at this level of variation being significant. However, there were intra-seasonal changes in expression of both Cry proteins and differences in expression between plant structures and between cultivars for both Cry proteins. Further work is needed to establish if this variation in Cry protein content in Bollgard II cotton affects the control of Helicoverpa spp. in the field or whether plant-physiological and pest-behavioural factors underlie the occasional occurrence of Helicoverpa larval survival on Bollgard II cotton.     

Feeding disruption tests for monitoring the frequency of larval lepidopteran resistance to Cry1Ac, Cry1F and Cry1Ab

An alternative to traditional larval lepidopteran resistance-monitoring bioassays was developed. Feeding disruption tests were developed for detecting insects resistant to three Bacillus thuringiensis (Bt) proteins: Cry1Ac, Cry1F and Cry1Ab. The assays rely on a diagnostic dose of Bt toxin in 100-μl hydratable meal pads of artificial diet containing blue indicator dye. The assay was formatted as a portable (palm-sized) plastic plate containing an array of 16 test wells, each containing a single hydratable meal pad with one insect added per well. The diagnostic dose was the concentration of Bt in meal pad rehydration solution that reduced 24 h dyed fecal production of Bt-susceptible neonates to ≤2 fecal pellets per larva. Bt-resistant neonates were able to consume the diagnostic dose of the insecticidal protein and produce >2 blue fecal pellets. The feces were distinctly visible on the white background of the feeding disruption test plate. Diagnostic doses were determined with lab-strain Bt-susceptible Heliothis virescens and Helicoverpa zea. For H. virescens, the diagnostic doses were 10, 20 and 15 μg/ml for Cry1Ac, Cry1F and Cry1Ab, respectively. For H. zea, the diagnostic doses were 40, 200 and 500 μg/ml, respectively. The assays were validated against a lab-strain of Bt-resistant H. virescens and with susceptible larval H. virescens collected as eggs from field-grown tobacco in North Carolina.     

Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation

Cry1Ab gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains (EHA105 and EHA101) contained Hpt and Cry1Ab genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and Cry1Ab genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T₂ progenies were analyzed for Cry1Ab and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive (80.94%), 62 plants were also Cry1Ab co-transformants (15.05% in total frequency), and 42 plants among the 272 T₂ progenies were Cry1Ab positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene. Southern blot analysis of five independent marker-free T₂ transgenic lines co-transformed from Zhejing 22 showed that Cry1Ab gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.     

转Bt基因水稻Cry1Ab杀虫蛋白在水稻土中的降解

室内研究了转 Bt基因水稻克螟稻 2号和华池B6 叶片中的Cry1Ab杀虫蛋白在稻田水稻土中的降解动态。分别将克螟稻 2号的粉碎叶片和华池B6 的整张叶片埋入 3种土壤 (即老黄筋泥田、青紫泥田和黄松田 )中 ,此后每隔 6～ 10d测定土壤或叶片中的Cry1Ab含量。克螟稻 2号粉碎叶片中的Cry1Ab均以处理后的前 36d,尤其是前 6d的降解较快 ,其中在青紫泥田中最快 ,黄松田中最慢。在处理后 6～ 36d内 ,不同土壤中的Cry1Ab残留量有显著差异 ,黄松田中的明显最高 ,老黄筋泥田中的次之 ,青紫泥田中的为最低 ;此后土壤间的残留量差异逐渐缩小 ,至处理后 78d差异已不明显。土壤淹水可显著加快华池B6 叶片中Cry1Ab的降解 ,且淹水后其降解动态在不同土壤之间十分相似 ;淹水对Cry1Ab降解的促进作用仅发生在前 12d之内 ,此后多数时间内残留量在淹水与非淹水处理间无显著差异。建立了各处理中Cry1Ab降解的动力学指数方程 ,相应地得出了其降解的半衰期。还讨论了土壤有机质和微生物等因子对土壤中Cry1Ab降解的影响。     

不同诱集作物对棉田刺吸性害虫及其天敌的生态作用比较

通过在棉田周围种植玉米、绿豆和向日葵等诱集作物,研究其对棉田蓟马、盲蝽的诱集效果和对草蛉、龟纹瓢虫和异色瓢虫等自然天敌的保护作用.试验结果表明,棉田四周种植这3种诱集作物,对棉田蓟马和盲蝽的诱集效果不明显,但对棉田草蛉、龟纹瓢虫和异色瓢虫的保护增殖作用非常明显.     

转基因抗草甘膦抗虫棉田主要害虫及其天敌的种群动态

 2010年在田间小区研究了转基因抗草甘膦抗虫棉田主要害虫和天敌的种群动态。结果表明,抗草甘膦抗虫棉不能减少田间棉铃虫的落卵量,但对棉铃虫有良好的控制作用。2、3代棉铃虫发生期,抗草甘膦抗虫棉田平均百株落卵量分别比Bt棉田增加12.5%和74.3%,比常规棉田增加4.6%和65.7%;棉铃虫平均百株幼虫数量少于常规棉田,但多于Bt棉田。棉盲蝽平均百株数量比Bt棉田和常规棉田分别减少36.1%和66.0%。抗草甘膦抗虫棉对棉蚜、棉叶螨和棉粉虱没有控制作用,和常规棉相比,田间发生量差异不显著,因此,仍需加强田间虫情监测,采取综合防治措施控制其为害。同时发现转基因抗草甘膦抗虫棉对草蛉、龟纹瓢虫、蜘蛛、小花蝽、寄生蜂等棉田主要天敌的种群数量均无显著影响。     

不同药剂对麦蚜的防治作用及对麦田天敌昆虫的影响

为了明确杀蚜药剂对麦蚜的防治效果、适宜的防治时期、对生态环境安全性和粮食作物食品安全性的影响,对4.5%氯氰菊酯、40%氧化乐果、40%毒死蜱、3%啶虫脒、10%吡虫啉等9种防治麦蚜的药剂进行了筛选,发现各药剂对麦蚜的防效较好,喷药后7 d防效均在90%以上.9种农药在喷施后的3 d内,对麦蚜的主要天敌蚜茧蜂和瓢虫影响较大,两种天敌数量锐减,但到7 d时各处理蚜茧蜂数量均有回升,其中不同剂型的吡虫啉、啶虫脒和鲁植顺克处理的蚜茧蜂数量同对照相差不大.由于瓢虫本身数量较少,而且喷药期内麦田瓢虫种群数量呈下降趋势,因此,种群数量回升不明显.在5月3日、5月8日、5月13日用4.5%高效氯氰菊酯、40%氧化乐果、5%蚜螨虫净超微乳油、20%吡虫啉可湿性液剂四种药剂进行了不同时期防治麦蚜农药试验,表明在麦蚜发生初盛期喷施杀蚜剂可以有效防治麦蚜.各农药处理小麦收获晒干后的农药残留量均在国家安全标准之内.根据试验结果,建议在麦蚜发生初盛期喷施吡虫啉或0.7%阿维.BTWP,可以减少喷药次数,提高作物产量,同时保护田间生态环境.