Transgenic cry1Ab/vip3H+epsps Rice with Insect and Herbicide Resistance Acted No Adverse Impacts on the Population Growth of a Non-Target Herbivore,the White-Backed Planthopper,Under Laboratory and Field Conditions

Numerous Bt rice lines expressing Cry protein derived from Bacillus thuringiensis Berliner (Bt) have been developed since 1989. However, the potential risks posed by Bt rice on non-target organisms still remain debate. The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the most economically important insect pests of rice in Asian countries and also one of the main non-target herbivores of transgenic rice. In the current study, impacts of transgenic cry1Ab/vip3H+epsps rice (G6H1) with both insect and herbicide resistance on WBPH were evaluated to ascertain whether this transgenic rice line had potential risks for this sap-sucking pest under laboratory and field conditions. The laboratory results showed that no significant difference in egg developmental duration, nymphal survival rate and female fecundity was found for WBPH between G6H1 and its non-transgenic isoline (XS110). However, the development duration of nymphs was significantly shorter and female longevity significantly longer when WBPH fed on G6H1 by comparison with those on its control. To verify the results found in laboratory, a 3-yr field trial was conducted to monitor WBPH population using both the vacuum-suction machine and beat plate methods. Although the seasonal density of WBPH nymphs and total density of nymphs and adults were not significantly affected by transgenic rice regardless of the sampling methods, the seasonal density of WBPH adults in transgenic rice plots was slightly lower than that in the control when using the vacuum-suction machine. Based on these results both from laboratory and field, it is clear that our tested transgenic rice line will not lead higher population of WBPH. However, long-term field experiments to monitor the population dynamics of WPBH at large scale need to be conducted to confirm the present conclusions in future.     

转cry2Ab4和vip3Aa11基因烟草对小地老虎杀虫效果研究

小地老虎严重危害农业生产,通过转基因技术提高作物对小地老虎的杀虫效果是一条切实可行的途径。cry2Ab4和vip3Aa11是从苏云金芽胞杆菌（Bacillus thuringiensis,Bt）中克隆到的两个杀虫基因。分别构建了pCS2Ab和pCSvip3A两种植物表达载体,利用农杆菌介导法转化烟草（Nicotiana tabacum L.）NC89,并得到32株和35株阳性转基因植株。通过对其进行RT-PCR分析、Western杂交分子检测证实两种外源基因在烟草植株中可以正常表达。人工饲喂初龄小地老虎幼虫,对两种转基因植株进行生物活性检测,结果显示饲喂转基因叶片的小地老虎幼虫死亡率在82%以上,抗虫能力比对照显著提高,转vip3A植株抗虫效果优于转cry2Ab植株。     

安庆稻区两种稻飞虱年度间种群动态的研究

在安庆稻区,水稻的混作提供了褐飞虱和白背飞虱种群发生与发展的生活条件。综合１９９５～１９９７年３年的研究结果,白背飞虱在８月中旬进入虫口高峰,褐飞虱于９月中旬出现最大虫量。前者年发生４～５代,后者则为３～４代。不同世代的田间种群数量、增殖倍数以及年度间的种群动态有种的特点。文中还讨论了制约田间的种群数量变化的可能因子。     

抗虫基因cry1Ab／Ac在水稻杂交转育后代中的表达效应

以转入了Bt基因cry1Ab/Ac的水稻品系TT51为供体,与10个恢复系杂交。通过分子检测,并结合田间的抗虫性观察,筛选出农艺性状良好的转基因纯合株系。抗虫基因在所有的杂交组合后代中按照孟德尔规律遗传,但是不同遗传背景下的表达量有很大差异。 Cry1Ab/Ac蛋白含量维持原有水平的株系对螟虫表现出良好的抗性,蛋白含量降低的株系则受到轻微的危害。纯合株系的部分农艺性状发生变异,其程度与Cry1Ab/Ac蛋白含量有关。结果为利用杂交转育培育优良抗虫水稻提供依据。     

白背飞虱灯诱种群数量季节动态

自1979年以来,在广东省肇庆市设置诱虫灯监测白背飞虱Sogatella furcifera(Horvath．)的种群数量变化．结果表明：白背飞虱各年的始见日在3月或4月内,始盛日在4月或5月内．年发生动态为双峰型,即早稻和晚稻高峰,早稻高峰多在5月上旬至7月下旬之间,晚稻高峰多在9月上旬至10月中旬之间．全年高峰有些年份是早稻高峰,而有些年份则是晚稻高峰．末见日多在11月下旬．根据24年发生高峰日的日诱获数量、旬平均诱获数量及全年的累计数量比较各年的发生严重程度,结果表明,20世纪70年代末至80年代初白背飞虱的发生严重,而90年代末,直至本世纪初发生较轻．     

转cry1C和cry2A基因Bt水稻对白背飞虱生长、发育及繁殖的继代影响

转Bt基因水稻对非靶标节肢动物的影响是转基因水稻安全性评价的重要组成部分。目前,已开展了大量关于转Bt基因水稻对非靶标害虫安全性评价的研究,但多限于1代,不能完全反映田间情况。试验以转Bt基因水稻T1C 19(含cry1C基因)和T2A 1(含cry2A基因)以及其亲本非Bt水稻MH63为材料,在室内条件下连续饲养白背飞虱4代,研究转Bt水稻对非靶标害虫白背飞虱生长发育和繁殖的继代效应,以明确T1C 19和T2A 1水稻对白背飞虱的潜在影响。结果表明,白背飞虱在3种水稻品种上连续取食4代以后,其生长发育(若虫发育历期、若虫存活率、雌成虫体重)和繁殖(产卵量和卵孵化率)指标均没有受到水稻品种的显著影响。可见,供试的T1C 19和T2A 1对白背飞虱无显著影响。     

cry1Ac基因启动子表达Vip3Aa蛋白特性分析

苏云金芽胞杆菌cry基因启动子常用于构建蛋白表达载体。为探讨苏云金芽胞杆菌cry基因启动子指导Vip3Aa蛋白表达情况及杀虫活性,以p UC19载体为基础,运用重叠引物PCR方法构建Vip3Aa11表达载体,并与由T7启动子指导的Vip3Aa11表达蛋白杀虫活性、抗胰蛋白酶稳定性比较,初步探索发酵条件。结果表明,cry1Ac启动子与T7启动子均在上清液中表达大小为88 ku Vip3Aa11蛋白,对甜菜夜蛾、棉铃虫杀虫活性差异不显著,cry1Ac基因启动子在37℃、48 h更适合Vip3Aa11蛋白的表达,为vip基因表达、功能验证及杀虫机理等研究提供新思路。     

转cry1Ab基因水稻对二化螟幼虫的致死和亚致死效应

测定了转cry1Ab基因水稻"克螟稻1号"对二化螟幼虫的致死和亚致死作用.结果表明,1龄、3龄、5龄幼虫持续取食"克螟稻1号"时,死亡率极高,5龄虽存活了较长时间,但不能正常化蛹.而当取食"克螟稻1号"一段时间后,改以亲本对照"秀水11"为食时,除在初期死亡较多外,死亡率不随时间而显著上升,能存活至下一龄期,部分还可化蛹,且幼虫总的历期与对照组差异不显著.幼虫取食一定时间的"克螟稻1号"后改食对照时,除1龄外,3龄和5龄幼虫经处理后所得的蛹均能羽化,其中3龄幼虫经处理后所得的成虫体重和体长与对照组差异不显著,而5龄幼虫经处理后所得的成虫体重和体长均显著低于对照组,但羽化率和产卵量并不显著低于对照组.     

Geographical Distribution of Bacillus thuringiensis Strains and vip3 Genes in Different Climatic Zones in China

Vegetative Insecticidal Proteins(VIPs),a large family of insecticidal proteins,are produced from Bacillus thuringiensis (Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity.This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical (Hainan Province), subtropical (Guangxi Province) and temperate zones (Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes,while vip3 genes were not expressed from temperate zone in Heilongjiang Province.Restriction Fragment Length Polymorphism(RFLP)was used to identify different types of vip 3 genes that were within the same family and three full-length vip3 genes were isolated.The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E.coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects (Spodoptera exigua and Agrotis ipsilon)were performed.The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay.Taken together,the results indicated that the distribution of Bt strains and vip3 genes had regional preference.Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region.These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources.The new vip 3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.     

苏云金杆菌营养期杀虫蛋白基因vip3A的检测与杀虫活性测定

为获取高活性的野生Bt菌株,对从土壤中分离的99株Bt菌株进行了营养期杀虫蛋白的分子鉴定和生物活性测定。根据已知的vip3 A基因序列设计1对特异性引物,进行PCR鉴定,55株扩增得到1.2 kb的目的片段。对31株阳性菌株的营养期杀虫蛋白活性进行了初步测定,结果显示,Bt LS1和LS8菌株毒力最高,2菌株对2龄甜菜夜蛾的体重抑制率分别为91.14%和93.59%。选取Bt LS1和LS8菌株进行胞内外营养期蛋白杀虫活性测定,发现Bt LS1和LS8菌株对初孵甜菜夜蛾体重抑制率分别为45.1%和43.2%,对初孵棉铃虫的校正死亡率分别为(22.1±1.4)%和(55.3±3.7)%,对2龄棉铃虫的体重抑制率分别为(78.7±6.6)%和(50.4±2.4)%。