华北北部地区转Cry1A+CpTI和Cry1A基因棉田节肢动物多样性*

2000～2001年采用背负式机动吸虫器取样法在河北省廊坊研究了转(Cry1A CpTI)基因棉(Gossypium hirsutum)(SGK321)、转Cry1A棉(GKl2)和其受体棉(G．hirsutum)棉田的节肢动物总群落、害虫亚群落、天敌亚群落和中性昆虫亚群落的多样性。采用SHANNON信息多样性指数分析显示,转抗虫基因棉的害虫多样性和天敌亚群落多样性与普通棉施药和非施药处理没有显著差异,但由于在棉花生长中、后期转抗虫基因棉处理的中性昆虫多样性显著高于普通棉施药处理,导致同期转基因棉的节肢动物总群落多样性明显高于普通棉的施药防治棉铃虫处理。表明转抗虫基因棉由于减少棉铃虫防治用药量,而显著提高了棉田中、后期生态系统节肢物的群落多样性,有利于生态系统的稳定和害虫的综合治理。     

不同生境对抗虫棉绿盲蝽及其天敌发生动态的影响

于2005-2007年采用田间调查方法对不同生境抗虫棉田内绿盲蝽及其天敌的发生动态进行了研 究, 试验设10种处理： 远离果园棉田、紧靠果园棉田, 密度为54 000株·hm^-2的均行距棉田、密度为45 000株·hm^-2的大小行距棉田, 合理化控棉田、不化控棉田, 纯作棉田和插种葵花棉田, 转双价基因抗虫棉田和转单价基因抗虫棉田。结果表明, 不同生境抗虫棉田内绿盲蝽天敌的种类基本相同, 主要为龟纹瓢虫、七星瓢虫、中华草蛉、大草蛉、华姬蝽、小花蝽、T-纹豹蛛、三突花蛛、草间小黑蛛等。不同生境抗虫棉田内绿盲蝽及其天敌种群数量差异明显。远离果园、密度为45 000株·hm^-2的大小行距、合理化控能减轻转基因抗虫棉田绿盲蝽的发生, 但不利于绿盲蝽天敌的发生;种植转双价基因抗虫棉能减轻绿盲蝽的发生且对绿盲蝽天敌影响不大, 插种葵花既能增加棉田绿盲蝽天敌的数量, 又能减轻绿盲蝽的发生。因此, 转双价基因抗虫棉田插种葵花, 并远离果园、合理化控、合理密度并大小行距种植是保益灭害控制绿盲蝽的有效方法。     

不同生境对抗虫棉绿盲蝽及其天敌发生动态的影响

于2005～2007年采用田间调查方法对不同生境抗虫棉田内绿盲蝽及其天敌的发生动态进行了研究, 试验设10种处理: 远离果园棉田、紧靠果园棉田, 密度为54 000株·hm-2的均行距棉田、密度为45 000株·hm-2的大小行距棉田, 合理化控棉田、不化控棉田, 纯作棉田和插种葵花棉田, 转双价基因抗虫棉田和转单价基因抗虫棉田.结果表明, 不同生境抗虫棉田内绿盲蝽天敌的种类基本相同, 主要为龟纹瓢虫、七星瓢虫、中华草蛉、大草蛉、华姬蝽、小花蝽、T-纹豹蛛、三突花蛛、草间小黑蛛等.不同生境抗虫棉田内绿盲蝽及其天敌种群数量差异明显.远离果园、密度为45 000株·hm-2的大小行距、合理化控能减轻转基因抗虫棉田绿盲蝽的发生, 但不利于绿盲蝽天敌的发生;种植转双价基因抗虫棉能减轻绿盲蝽的发生且对绿盲蝽天敌影响不大, 插种葵花既能增加棉田绿盲蝽天敌的数量, 又能减轻绿盲蝽的发生.因此, 转双价基因抗虫棉田插种葵花, 并远离果园、合理化控、合理密度并大小行距种植是保益灭害控制绿盲蝽的有效方法.     

转Cry1Ac和Cry1A+CpTI基因棉花粉对柞蚕生长发育影响的评价*

柞蚕(Antheraea pemyi)是中国华北和东北地区大面积人工野外饲养的重要经济昆虫,以天然柞林形成的蚕场与棉花常处于同一生态系统中。采用田间花粉沉积量测定结合室内生物测定的方法评价了转Cry1Ac基因和Cry1A CpTI基因棉(Gossypium hirsutum)花粉对柞蚕生长发育的生态风险。花粉沉积量测定表明,棉田内1m至棉田外1m的范围内,棉花花粉5d累积密度低于200粒／cm^2。用转基因抗虫棉花粉处理(花粉密度约l000粒／cm^2和10000粒／cm^2)的辽东栎(Quercus liaotungensis)叶片分别饲养蚁蚕至3龄末,结果显示,与非转基因棉花粉处理相比1～3龄期柞蚕的死亡率、发育历期、以及食物利用率、食物转化率、相对代谢速率、相对取食量和近似消化率等营养指标均无显著差别。表明两种供试转基因抗虫棉的种植不会对柞蚕的生长发育带来显著不利影响。     

淮北棉田害虫与捕食性天敌群落动态变化研究

对淮北棉田害虫和捕食性天敌群落动态变化进行了研究。田间系统调查共发现害虫35种,捕食性天敌24种;害虫优势种为棉蚜、烟粉虱、棉叶螨、棉盲蝽;捕食性天敌优势种主要为四点亮腹蛛、龟纹瓢虫、T-纹豹蛛、草间小黑蛛、八斑球腹蛛、华姬猎蝽、小花蝽、青翅隐翅虫。该区棉田害虫与捕食性天敌在棉株上的分布特点为:四点亮腹蛛、T-纹豹蛛、草间小黑蛛、八斑球腹蛛、小花蝽、青翅隐翅虫为下部>中部>上部;烟粉虱、棉盲蝽为上部>中部>下部;华姬猎蝽7月30日前为上部>下部>中部,8月9日后为下部>上部>中部;棉蚜在6月20日前为上部>下部>中部,在6月30日后为下部>上部>中部;棉叶螨前期主要分布在下部,后期向上转移;龟纹瓢虫在7月10日前为上部>中部>下部,7月20日之后为下部>上部>中部。群落均匀度在棉花生长发育前期较低,中期逐渐上升,后期又逐渐下降;集中度的变化趋势与均匀度相反;多样性在棉花生育中期最高。以物种为单位和以功能团为单位的群落多样性指数和均匀度指数呈高度正相关,相关系数(r)为0.9904。     

转Cry1Ab/Ac基因水稻对稻田底栖动物群落的影响

转Bt基因抗虫水稻的成功研发,为有效控制鳞翅目害虫对水稻的危害提供了一条最为有效、经济的途径。稻田底栖动物群落是转Bt基因抗虫水稻的重要非靶标生物。为了解转Bt基因水稻对稻田底栖动物群落的安全性,以转Cry1Ab/Ac基因抗虫水稻‘华恢1号’(简写为HH1)为材料,以其非转基因亲本‘明恢63’(简写为MH63)为对照,以稻田底栖动物群落为指示生物,运用群落学方法,通过连续2年在江西南昌的大田试验,研究了转Cry1Ab/Ac基因抗虫水稻对稻田底栖动物群落的影响。结果显示:2012年在HH1与MH63的稻田中分别采集到底栖动物22种与25种,其中两生境中相同物种19种,两群落物种组成相似度为0.808 5,有7种优势种,优势种相似度为0.833 3。2013年在HH1与MH63稻田中分别采集到底栖动物26种与28种,两类稻田相同底栖动物22种,两群落物种组成相似度为0.814 8,有6种优势种,优势种相似度为1.000 0。2012年与2013年群落总体分析与时间动态分析均显示,两类稻田中底栖动物的物种丰富度、个体数量、多样性指数、均匀性指数及优势集中性指数变化趋势相似,且均无显著差异。结果表明:连续种植2年转Cry1Ab/Ac基因水稻HH1对南昌稻田底栖动物群落无明显的负作用。     

转单价及双价基因抗虫棉田棉铃虫发生动态初报

试验研究转单价及双价基因抗虫棉田棉铃虫的发生动态结果表明,转单价及双价基因抗虫棉的棉叶、蕾、铃被害率均显著低于常规棉,抗虫棉田第2代、3代棉铃虫幼虫发生数量均很少,第4代虽有一定数量发生但低于常规棉棉田。第2代、3代和4代棉铃虫发生期间,转单价及双价基因抗虫棉田其幼虫平均数量及棉叶、蕾、铃被害率均未达显著差异水平,表明在田间转单价与双价基因抗虫棉对棉铃虫的抗性差异不明显。     

不同类型棉田昆虫群落结构研究

运用群落生态学原理和方法,于2007年6月26日至8月20日系统调查了平作春棉田和夏棉田主要害虫的种群数量动态,并利用相对丰富度、优势度指数、优势集中性指数、多样性指数和均匀度分析了其群落结构和时间动态.结果表明:两类棉田昆虫群落组成较为丰富,由10目35科48种组成.其中春棉田昆虫群落为9目30科39种,夏棉田昆虫群落为10目30科43种;优势种是棉蚜(Aphis gossypii Glover.)、烟粉虱(Bemisia tabaci Gennadius)和朱砂叶螨[Tetranychus cinnarinus(J(Bois)].在6月底至7月上旬春棉田优势种主要有朱砂叶螨,7月初数量达最多;两类棉田在7月中旬以后优势种均为棉蚜,7月底至8月中旬优势种均为烟粉虱.两类棉田昆虫群落的总个体数变化较大,但春棉田中昆虫群落和害虫亚群落总个体数始终高于夏棉田;春棉田害虫的相对丰富度较大,而夏棉田天敌的相对丰富度较大;两类棉田以寄生性天敌群落相似系数最高(0.949 4),植食性害虫群落最低(0.439 4);春棉田昆虫群落多样性指数最高为1.895 3,夏棉田昆虫群落多样性指数最高为2.095 1,均匀度的变化趋势与多样性指数变化基本一致,而优势集中性指数的变化趋势与多样性指数和均匀度的变化正好相反,优势集中性指数越大,多样性指数和均匀度越小.     

麦田害虫与天敌群落动态变化研究

2008年3月15日至5月29日系统调查了冬小麦田害虫与天敌的种群数量动态,并利用相对丰富度、优势度指数、优势集中性指数、多样性指数和均匀度分析了害虫与天敌群落动态变化及时间生态位。结果表明:麦田昆虫群落组成较为丰富,由11目39科59种昆虫组成;优势种为麦长管蚜(Macrosiphum avenae)、麦圆叶爪螨(Penthaleus major)、龟纹瓢虫(Propylea japonica)和麦蚜茧蜂(Ephedrus plagiator)。群落的总个体数和丰富度随调查时间而变化;多样性指数与均匀度指数在小麦生长前期较低,中期和后期较高,两者变化规律基本一致,而优势集中性指数变化趋势与其相反。在害虫种群中以棉铃虫(Helicoverpa armigera)种内竞争最为激烈,条斑叶蝉(Psammotettix striatus)的竞争最缓和;棉铃虫与麦长管蚜种间竞争最强,其次为麦圆叶爪螨与小麦叶蜂(Dolerus tritici)。在天敌种群中以七星瓢虫(Coccinella septempunctata)种内竞争最为激烈,麦蚜茧蜂的种内竞争最为缓和;龟纹瓢虫分别与七星瓢虫和异色瓢虫(Leis axyridis)在时间维度上的竞争较为激烈,而麦蚜茧蜂分别与龟纹瓢虫、七星瓢虫和异色瓢虫的竞争比较缓和。从害虫种群与天敌种群之间的重叠值看,异色瓢虫和七星瓢虫对棉铃虫的控制作用较强,而麦蚜茧蜂和龟纹瓢虫则对麦田主要害虫麦长管蚜、条斑叶蝉和小麦潜叶蝇(Phytomyza nigra)等的跟随和控制作用均较强。     

转基因水稻B2A68中抗虫蛋白Cry2Aa的表达特征和抗性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)基因的高效表达和稳定遗传对抗虫转基因水稻(Oryza sativa)的培育和商业化生产至关重要。转基因水稻B2A68是通过农杆菌介导法把双丙氨酰磷抗性基因(biplaphos resistance gene,Bar)和Bt杀虫晶体蛋白基因(insecticidal crystal proteins gene)Cry2Aa同时转入到恢复系D68中,培育出的抗除草剂、抗螟虫的两系早稻恢复系。本研究运用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测法研究了B2A68纯杂合基因型、不同组织及不同发育时期叶片中Cry2Aa蛋白的时空表达特征,并结合室内生测实验,揭示抗虫蛋白的表达水平、时期、部位与抗虫性的相关性。研究表明,转基因抗虫水稻B2A68纯合植株叶片中Cry2Aa蛋白表达量极显著高于杂合植株。乳熟期各器官中Cry2Aa蛋白含量为:叶片颖壳和脱壳籽粒茎秆和叶鞘(P0.05);腊熟期各器官中Cry2Aa蛋白含量为:叶片脱壳籽粒茎秆和颖壳(P0.05),但脱壳籽粒和叶鞘之间、叶鞘和茎秆之间、茎秆和颖壳之间的Cry2Aa蛋白含量差异不显著。从秧苗期到腊熟期的叶片中Cry2Aa蛋白含量表现为先升后降再升高的波动,且T_3、T_4两代波动趋势一致。二化螟幼虫(Chilo suppressalis)死亡率与乳熟期各器官中的Cry2Aa蛋白含量高度相关(r=0.837),稻纵卷叶螟幼虫(Cnaphalocrocis medinali)死亡率与各发育时期叶片中的Cry2Aa蛋白含量高度相关(r=0.988)。Cry2Aa蛋白含量较低的杂合植株叶片对稻纵卷叶螟幼虫的全部致死时间稍长,但不超过5 d。本研究结果对田间害虫的防治以及转基因水稻的育种都具有参考价值。     

苏云金芽孢杆菌Cry1Aa和Cry3A结构域编码区的融合

苏云金芽孢杆菌（Bacillus thuringiensis,Bt)毒素蛋白由3个不连续的结构域（domains)组成，其中结构域I为膜跨越区，与膜孔道形成有关，结构域Ⅱ，Ⅲ与受体结合有关。利用基因重组技术以鳞翅目昆虫具专一活性的毒素蛋白Cry1Aa与专一性有关的结构域编码区与对鞘翅目昆虫具专一活性的毒素蛋白Cry3A编码基因融合，构建成融合表达载体，为进一步构建Bt工程菌和Bt转基因植物奠定基础。     

Rapid digestion of Cry34Ab1 and Cry35Ab1 in simulated gastric fluid

Two genes were identified in Bacillus thuringiensis Berliner (Bt) that code for the proteins that comprise a Cry34Ab1/Cry35Ab1 binary insecticidal crystal protein. Maize, Zea mays L., plants have been transformed to express the Cry34Ab1/Cry35Ab1 proteins, and as a result, these plants are resistant to attack by western corn rootworm, Diabrotica virgifera virgifera LeConte, a major pest in the Midwestern corn-growing area of the U.S.A. As part of the safety assessment for the proteins, digestibility studies were conducted. Digestion experiments with both proteins demonstrated rapid degradation in simulated gastric fluid, comparable to other registered plant-incorporated protectants. Quantitative and qualitative approaches for determining digestibility are illustrated.     

Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens

Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.     

转基因高粱Cry1Ab蛋白含量的比较研究

通过农杆菌介导法将杀虫晶体蛋白基因cry1Ab导入高粱恢复系115中,共获得13个独立的转基因株系,26株转基因植株,转化率为5.1%;GUS活性、PCR、Southern和Western杂交分析表明,此基因已整合进高粱基因组并得到正确表达。利用ELISA试剂盒测定Cry1Ab蛋白含量,结果表明,不同转基因植株的Cry1Ab蛋白含量有明显差异,最高可达0.850 μg/g,占可溶性蛋白的0.016%,还有一些转基因植株中不能表达;同一转基因植株的不同组织中表达量有明显差异,其顺序为：叶>颖壳>籽粒>根>茎;不同部位的叶片也有差异,其顺序为：中部叶>基部叶>新叶。     

Mineralization of the Bacillus thuringiensis Cry1Ac endotoxin in soil

Although a number of studies have been done describing the fate of Bacillus thuringiensis insecticidal endotoxins in soil, there is conflicting information on the persistence of this class of insecticidal toxins. This is partly due to methodological limitations in many of the previous studies. In the experiments reported here, 14C-labeled B. thuringiensis Cry1Ac endotoxin was used to study its mineralization in soil incubated under controlled conditions. Fifty-nine percent of the radiolabeled Cry1Ac was recovered as 14CO2 at the end of the 20 day incubation period. The addition of 4.5% corn residues stimulated mineralization of [14C]Cry1Ac toxin, and mineralization of glucose was 3.6 times faster than that of the Cry1Ac toxin, indicating that the soil was microbiologically and metabolically active. Because only low mineralization (approximately 6%) of the radiolabeled toxin was observed in autoclaved soil, the current findings indicate that microbial processes play a major role in the dissipation of the Cry1Ac endotoxin in soil. The results of this study suggest that there may be limited risk of the bioaccumulation of Cry1Ac in soil due to the eventual release of this insecticidal toxin by Bt-protected crops.     

Influence of Cry1Ac toxin on mineralization and bioavailability of glyphosate in soil

The impact of transgenic plants containing Bacillus thuringiensis (Bt) toxin on soil processes has received recent attention. In these studies, we examined the influence of the lepidopterean Bt Cry1Ac toxin on mineralization and bioavailability of the herbicide glyphosate in two different soils. The addition of 0.25-1.0 microg g(-1) soil of purified Cry1Ac toxin did not significantly affect glyphosate mineralization and sorption in either a sandy loam or a sandy soil. In contrast, extractable glyphosate decreased over the 28 day incubation period in both soils. Our findings suggest that the reduction in the bioavailability of glyphosate was not influenced by the presence of Cry1Ac toxin but rather the results of aging or sorption processes. Results from this investigation suggest that the presence of moderate concentrations of Bt-derived Cry1Ac toxin would have no appreciable impact on processes controlling the fate of glyphosate in soils.     

Cry1Ab Adsorption and Transport in Humic Acid-Coated Geological Formation of Alumino-Silica Clays

Genetically modified agricultural products have been introduced to increase food supply by enhancing their resistance to pests and diseases, along with easily adapting to environmental conditions. Due to the modification of DNA, public objections are prevalent, including concerns on the impact on the ecosystem. In this research, adsorption and transport of Cry1Ab, a toxin exuded by the transgenic Bt maize in alumino-silica clays, were evaluated in laboratory columns under steady-state flow conditions. Since Cry1Ab fate and transport were very responsive to animal waste field applications, during which humic acids were released, Cry1Ab adsorption and transport in humic acid-coated alumino-silica clays were also investigated. Cry1Ab breakthrough curves were simulated using the convection-dispersion transport models. It was discovered that the humic acid coating increased Cry1Ab deposition during the transport. Based on analysis of the breakthrough curves, adsorption isotherms of Cry1Ab in alumino-silica clays were obtained and compared with those of batch experiments. The humic acid coating changed the bonding energy between Cry1Ab and the adsorption receptor sites on alumino-silica clay surfaces, thereby changing Cry1Ab partition between the aqueous phase and the solid phase.     

Bt晶体蛋白Cry1Ac放射免疫检测技术研究

通过苏云金芽孢杆菌(Bt)HD-73培养,提取Bt晶体蛋白Cry1Ac,经酶解后获得高抗虫活性蛋白。免疫新西兰白兔得到高纯度的Bt晶体蛋白1Ac抗体,用125I标记抗原,研究和建立了Bt晶体蛋白Cry1Ac的放射免疫检测技术,该试剂盒用磁性微粒作分离剂,不需离心即可分离,简化了测定步骤,检测结果达到国外同类产品(酶联免疫试剂盒)的水平。     

Rapid degradation of the Cry1F insecticidal crystal protein in soil

The gene for the core Cry1F insecticidal crystal protein (ICP) from Bacillus thuringiensis Berliner (Bt) has been incorporated into the genome of maize plants, Zea mays L. Plants expressing this ICP are protected from attack by various Lepidopteran pests including the European corn borer, Ostrinia nubilalis (Hübner). The stability of the Cry1F ICP in soil was assessed in a laboratory study designed to determine the persistence of the active protein residue in soil over time, using insect bioassay as the analytical quantification method. The GI(50) (concentration estimated to inhibit growth by 50%) rose at each consecutive incubation interval, indicating a consistent decline in Cry1F activity over time. The residue data were poorly described by a first-order model when fit to either the full data or a truncated data set where the last interval (28 days) was excluded. Data were well described by a shift-log model, and this model predicted DT(50) (time until 50% decay) and DT(90) (time until 90% decay) values of 0.6 and 6.9 days, respectively. This rapid degradation rate was consistent with other Bt proteins evaluated in our laboratory.