用Envirologix Cry1Ab/Cry1Ac试剂盒快速测定转基因水稻Bt杀虫蛋白含量的研究

 用 Envirologix Cry1Ab/Cry1Ac平板试剂盒检测了两个转 cry1Ab的水稻品系克螟稻 1号 ( KMD1)、克螟稻 2号 ( KMD2 ) ,及未转化的对照品种秀水 11米粒中的 Bt杀虫蛋白含量。结果表明 ,该试剂盒标样制作的标准曲线相关系数为 0 .985～ 0 .998,在 0 .0 5或 0 .0 1水平上显著。同批次试剂盒的不同试验及不同批次试剂盒的测定值均差异不显著。最低检测剂量达 0 .5 ng/g,每次测试时间比 Western dot blotting方法缩短约 2 h。试验表明 ,该试剂盒是定量检测转基因水稻 Bt毒蛋白表达的一种理想产品     

影响Bt稻离体叶中Cry1Ab杀虫蛋白降解的环境因子研究

 【目的】研究转Bt基因水稻表达的外源Bt杀虫蛋白在土壤中的环境去向，以便进行转BT基因水稻的生态风险性评价。【方法】室内采用ELISA法，研究了转Bt基因水稻克螟稻2号（KMD2）粉碎叶片中Cry1Ab杀虫蛋白在3种水稻土，即青紫泥田、黄筋泥田和黄松田土中不同土壤含水量、土壤pH值和温度条件下的降解动态。【结果】供试叶片粉中Cry1Ab蛋白在3种水稻土中的降解动态均可用一级化学反应动力学指数方程来拟合，降解半消减期t0.5为1.8～4.0 d。【结论】土壤含水量、pH和温度对Cry1Ab蛋白降解速率均有一定影响，但pH和温度的影响更为明显。通常pH较低的酸性土壤和低温不利于土壤中Cry1Ab蛋白的降解,特别是在酸性黄松田中降解最慢。     

转Bt基因抗虫玉米根茬和根际土壤中Cry1Ab杀虫蛋白的田间降解动态

【目的】研究转cry1Ab杀虫蛋白基因玉米收获后玉米根茬及其根际土壤中Cry1Ab杀虫蛋白的降解动态,比较两种Bt玉米根茬和根际土壤中Cry1Ab杀虫蛋白的降解速度。【方法】以两种表达Cry1Ab杀虫蛋白的Bt抗虫玉米MON810和Bt11为材料,采用ELISA方法测定玉米收获后根茬残体和根际土壤中Cry1Ab杀虫蛋白的田间降解动态。【结果】转Bt基因玉米根茬残体和根际土壤中杀虫蛋白是逐渐降解的,Bt玉米MON810根茬中Cry1Ab杀虫蛋白含量较高,降解的速度也较慢,收获后8个月时还不能完全降解;Bt玉米Bt11根茬中Cry1Ab杀虫蛋白含量较低,降解速度比MON810根茬中Cry1Ab杀虫蛋白降解速度快,到7个月时已检测不到Cry1Ab杀虫蛋白。Bt玉米MON810根际土壤中Cry1Ab杀虫蛋白的降解较Bt11的慢,MON810和Bt11根际土壤分别在8个月和7个月时检测不到Cry1Ab杀虫蛋白。【结论】种植过Bt11和MON810抗虫玉米的田块,在第二年春播农作物已经出土时,其根茬和根际土壤中残留的Cry1Ab杀虫蛋白尚不能完全降解,还有少量残留。     

“克螟稻”后代cry1Ab基因表达特性及其对二化螟抗性的研究

 以不同世代的“克螟稻”为试验材料 ,在田间自然条件下研究了 cry1Ab基因在“克螟稻”后代中的表达特性及其对二化螟的抗性。结果表明 ,cry1Ab基因在“克螟稻”后代中的表达表现出了一定的时空特性。在时间上 ,主茎叶片中 Cry1Ab蛋白含量在孕穗期为最高 ,在抽穗期为最低 ,除苗期外 ,在其它各个时期 Cry1Ab蛋白含量差异均不显著 ;在空间上 ,主茎、一次分蘖和二次分蘖各部位 Cry1Ab蛋白含量均不同 ,但均以茎、叶、叶鞘中表达量较高 ,根中表达量则相对较低。在同一主茎或分蘖上 ,不同叶位、叶鞘及茎段 Cry1Ab蛋白含量均不同。对二化螟抗性的室内生物测定及田间自然条件下抗性的调查表明 ,不同世代的“克螟稻”后代均对二化螟具有极强的抗性 ,对二化螟初孵幼虫的致死率可达 10 0 %     

转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白田间降解动态

 【目的】研究间苗后留在田间地表的转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白的降解规律，比较两种Bt玉米幼苗残体中Cry1Ab杀虫蛋白的降解速度。【方法】以两种表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米MON810和Bt11为材料，采用ELISA方法测定各取样时期中幼苗残体中Cry1Ab杀虫蛋白残留量。【结果】转Bt基因玉米幼苗残体中杀虫蛋白降解是逐渐的，且降解速度较快，到50 d时幼苗残体已经完全腐烂，Bt11幼苗残体中的杀虫蛋白已经完全降解，在MON810中还能检测到微量的杀虫蛋白。两种转基因玉米幼苗残体中的Bt杀虫蛋白的初始含量差异不显著，但在同一时间段的Bt杀虫蛋白降解速度存在差异均显著，在30 d前MON810幼苗残体中Bt杀虫蛋白降解速度比Bt11降解的快，30d后，则降解趋势相反，到50 d取样结束时MON810和Bt11分别降解了初始含量的99.81%和100%。【结论】两种转Bt基因玉米间苗后留在田间的幼苗残体中的Cry1Ab杀虫蛋白降解速度不同，在50 d完全腐烂时，其中的杀虫蛋白完全降解或仅有微量残留。     

转Bt基因玉米幼苗残体中Ory1Ab杀虫蛋白田间降解动态

【目的】研究间苗后留在田间地表的转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白的降解规律,比较两种Bt玉米幼苗残体中Cry1Ab杀虫蛋白的降解速度。【方法】以两种表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米MON810和Bt11为材料,采用ELISA方法测定各取样时期中幼苗残体中Cry1Ab杀虫蛋白残留量。【结果】转Bt基因玉米幼苗残体中杀虫蛋白降解是逐渐的,且降解速度较快,到50d时幼苗残体已经完全腐烂,Bt11幼苗残体中的杀虫蛋白已经完全降解,在MON810中还能检测到微量的杀虫蛋白。两种转基因玉米幼苗残体中的Bt杀虫蛋白的初始含量差异不显著,但在同一时间段的Bt杀虫蛋白降解速度存在差异均显著,在30d前MON810幼苗残体中Bt杀虫蛋白降解速度比Bt11降解的快,30d后,则降解趋势相反,到50d取样结束时MON810和Bt11分别降解了初始含量的99.81%和100%。【结论】两种转Bt基因玉米间苗后留在田间的幼苗残体中的Cry1Ab杀虫蛋白降解速度不同,在50d完全腐烂时,其中的杀虫蛋白完全降解或仅有微量残留。     

Bt杀虫蛋白在亚洲玉米螟幼虫体内组织中的分布

采用ELISA法检测了亚洲玉米螟取食转基因玉米后,Bt杀虫蛋白(Cry1Ab杀虫蛋白)在幼虫体内组织中的分布情况.结果表明,中肠和血淋巴中Bt杀虫蛋白含量最高,分别为267 ng·g-1和417 ng·g-1,丝腺、脂肪体、马氏管的含量较低,生殖器官中未检测到Bt杀虫蛋白;幼虫取食转基因玉米3d后改喂普通玉米的亚洲玉米螟发育成的蛹、成虫及卵中不含Bt杀虫蛋白.     

Bt玉米杀虫蛋白含量的时空表达及对亚洲玉米螟的杀虫效果

 利用ELISA方法对2个转Cry1Ab基因抗虫玉米品种MON810和Bt11在不同生育期、不同组织器官中杀虫蛋白的表达量进行了分析,利用室内生测方法研究了不同组织器官对亚洲玉米螟初孵幼虫杀虫效果,并分析了杀虫蛋白表达量与杀虫效果的相关性。结果表明,Cry1Ab杀虫蛋白在Bt玉米MON810和Bt11的不同生育期和组织中的表达量呈明显的时空动态变化,以营养生长阶段的心叶组织表达最高,分别为1880.6和1473.1ng·g-1,生殖生长阶段的花粉含量最低,分别为52.3和73.3ng·g-1。随着叶片的生长,     

转基因植物中Bt Cry2Ab蛋白的抗原表位特征预测

[目的]通过生物信息学方法了解转基因作物中Bt Cry2Ab蛋白的二级结构、三级结构、B细胞抗原表位及T细胞抗原表位等结构特征,为今后的抗体设计奠定基础。[方法]以在NCBI数据库中获得Cry2Ab蛋白序列为材料,应用DNAStar中的多种方法预测其B细胞表位,并通过在线预测软件NetMHCII 2.2分析Cry2Ab蛋白与MHC-II类分子的结合能力,从而预测其T细胞抗原表位。[结果]对Cry2Ab蛋白B细胞抗原表位的预测表明,Cry2Ab蛋白的第208～215区域是潜在的B细胞抗原表位;对Cry2Ab蛋白与MHC-II类分子结合能力的分析表明,Cry2Ab蛋白的第177～185区域、299～307区域和255～263区域是潜在的T细胞抗原表位,暗示含有HLA-DRB10101和HLA-DRB10701等位基因的人群对Cry2Ab蛋白更敏感。[结论]该研究有助于深入了解Cry2Ab蛋白的生物学特征,为完善转基因食品过敏原性的评估方法提供新线索。     

Cry1Ia和Cry2Ab融合蛋白的表达

[目的]苏云金芽孢杆菌表达的杀虫蛋白Cry1Ia和Cry2Ab对鳞翅目和鞘翅目昆虫具有较好的杀虫活性。为充分利用已有Cry蛋白,本文构建了两者的融合蛋白,并对融合蛋白的原核表达进行检测。[方法]本文将两个基因的编码基因,按照两种先后顺序拼接起来,获得cry1Ia-cry2Ab融合基因和cry2Ab-cry1Ia融合基因。另外,将Cry1Ia蛋白N端第一个α-螺旋(73个氨基酸)的编码序列删除后,接入cry2Ab基因5'端,获得第三个融合基因cry1IaD73-cry2Ab。将3个基因分别插入pHT304载体,并由cry1Ac基因的启动子和终止子调控其在Bt细胞中表达。[结果]SDS-PAGE结果显示,Cry1Ia-Cry2Ab、Cry2Ab-Cry1Ia和Cry1IaD73-Cry2Ab三个融合蛋白均能够在Bt细胞中表达,其测定分子量与预期大小一致,分别约152kDa、152kDa和144kDa。蛋白印迹法分析发现,在表达过程中,三种融合蛋白均有一定比例的降解。另外,不同温度对融合蛋白的表达也有不同程度的影响。[结论]本研究构建了3种融合杀虫基因并在Bt细胞中成功表达,为进一步解析这类融合蛋白的杀虫活性和机理奠定了基础。     

转Bt基因水稻的碳代谢特性

【目的】评价外源Bt基因导入对水稻碳代谢特性的影响。【方法】以转Bt基因水稻克螟稻1号及其非转基因亲本秀水11,以及克螟稻1号与3个杂交水稻恢复系明恢63、R3027和99亚162杂交和连续回交而育成的农艺性状和抗虫性稳定品系的3对近等基因系为材料,对叶绿素与可溶性糖含量、RuBP 羧化酶、蔗糖合成酶及蔗糖磷酸合成酶活性、干物质及有机碳积累量等碳代谢指标进行了测定。【结果】分蘖盛期克螟稻1号的叶绿素a含量、可溶性糖含量、干物质和有机碳含量均极显著低于秀水11（P＜0.01）,叶绿素b含量、蔗糖合成酶和蔗糖磷酸合成酶活性显著低于秀水11（P＜0.05）,但其它3对Bt水稻与非Bt水稻间所有生理指标均无显著差异;齐穗期克螟稻1号蔗糖磷酸合成酶活性显著高于秀水11,而干物质和有机碳积累量则显著低于秀水11;BtR3027蔗糖合成酶活性显著低于R3027,而Bt99亚162可溶性糖含量和蔗糖磷酸合成酶活性则显著高于99亚162;成熟期所有Bt水稻与各自非Bt水稻间干物质重和有机碳积累量差异均不显著。【结论】克螟稻1号大多数碳代谢指标的显著变化主要是由无性系变异引起的;3对近等基因系中Bt基因对个别碳代谢指标的影响是短暂的,且与Bt基因所处的遗传背景有关。     

Impacts of Environmental Factors on Degradation of Cry1Ab Insecticidal Protein in Leaf-Blade Powders of Transgenic Bt Rice

The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of Cry1Ab protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry1Ab were measured using enzyme-linked immunosorbent assay (ELISA). The degradation rate of Cry1Ab was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of Cry1Ab, but the effects of soil pH and temperature were relatively greater. In general,Cry 1 Ab degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.     

Impacts of Environmental Factors on Degradation of CrylAb Insecticidal Protein in Leaf-Blade Powders of Transgenic Bt Rice

The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of CrylAb protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry lAb were measured using enzyme-linked immunosorbent assay （ELISA）. The degradation rate of CrylAb was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of CrylAb, but the effects of soil pH and temperature were relatively greater. In general, CrylAb degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.     

Studies on Rapid Quantitative Analysis of Bt Toxin by Using Envirologix Kits in Transgenic Rice

Investigations were done on the usefulness of Envirologix Cry1Ab/Cry1Ac Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2), transformed with a cry1Ab gene, and their parental variety, cv.Xiushui 11, were used as positive and negative samples. Results showed that the correlation coefficients as high as 0. 985 - 0. 998, significant at probability level of 0.05 or 0.01, were obtained for linear regression equations by using the appended calibrators of the kits. No significant differences were detected for values of same rice sample obtained from different trials or by using different lots of the product (kit). The detectable Bt toxin content by this method could be as low as 0.5 ng/g. The Envirologix Kit could be useful for rapid quantitative detection of Bt toxin in rice grains because of its preciseness, simplicity and time saving.     

Thrips-mediated impacts from transgenic rice expressing Cry1Ab on ecological fitness of non-target predator Orius tantilus(Hemiptera:Anthocoridae)

Various rice lines have been genetically modified with genes from Bacillus thuringiensis(Bt) to continuously produce Bt insecticidal proteins against lepidopteran larvae. The Bt insecticidal protein constantly expresses in the plants to create an opportunity for non-target herbivores to acquire and convey the protein to their predators or parasitoids across trophic levels. This paper evaluates the effects of Bt rice(namely, Kemindao 1(KMD1) and Kemindao 2(KMD2)) expressing Cry1 Ab as compared to its non-Bt control line, Xiushui 11 on non-target predator Orius tantilus(a generalist predatory anthocorid of thrips) under laboratory and field conditions. To measure several biological parameters such as total nymphal duration and fecundity of this bug, it was reared on thrips and pollens of KMD1 and KMD2 as compared to their control under laboratory conditions. By comparison with the control, Bt rice did not significantly affect main life-history characteristics(total nymphal duration, female adult longevity, oviposition period and fecundity) of this anthocorid preying on Bt rice-fed thrips along with Bt rice pollens, except that the fecundity of this predator for KMD1 was distinctly lower as compared with KMD2 or the control. Enzyme-linked immunosorbent assay(ELISA) results showed that no Cry1 Ab protein was detected in this predator fed on thrips or rice pollen from Bt rice but was in Bt rice pollens. With the beat plate, plastic bag and color trap sampling methods, two-year field monitoring of O. tantilus abundance demonstrated that Bt rice had no significant detrimental effects on the population dynamics and seasonal average densities of this predatory anthocorid as compared with the control. Thus, it is suggested that growing our tested Bt rice(KMD1 and KMD2) producing Cry1 Ab will pose a negligible risk to the anthocorid, O. tantilus.     

转Bt基因水稻生长期几种重要成分含量的变化研究

 对Bt水稻汕优63及其非转基因亲本汕优63不同生长期几种重要成分的含量进行了分析，以期为转Bt基因水稻的生态学和安全性评价提供基础代谢方面的依据。结果表明，在抽穗期，Bt水稻和非Bt水稻茎秆和叶片中大部分氨基酸的含量差异显著或极显著；在大部分生长期，Bt水稻和非Bt水稻氮、磷和硅的含量差异显著或极显著，但它们的变化趋势基本一致，氮和磷的含量在拔节期之后均呈下降趋势，而硅的含量则随水稻的生长发育呈上升趋势；在水稻生长前期，Bt水稻可溶性糖和还原糖的含量比非Bt水稻低，但在后期比非Bt水稻高，且差异均显著或极显著。     

水稻分蘖期生理耗水量研究

[目的]研究水稻分蘖期生理耗水量,以期更加高效地节约灌溉水资源。[方法]采用室内盆栽方法,对水稻进行控水灌溉,根据每天每试验盆重量变化分析水稻生理耗水量。[结果]水稻分蘖前期耗水量不断增加,到分蘖后期逐渐降低,水稻分蘖前期平均每株每天耗水193.75 g,水稻分蘖后期平均每株每天耗水260.92 g。每盆栽2株水稻耗水量高于每盆栽1株水稻耗水量33.4%。[结论]在水稻分蘖末期应控制灌溉水量;水稻种植越密,分蘖期生理耗水量小,其分蘖数相应减少。     

转Bt(Cry1Ab)基因对水稻光合特性及光合产物积累的影响

 【目的】明确外源Bt基因插入对水稻倒1叶光合速率及光合作用相关生理特性的影响。【方法】以转Bt基因水稻及亲本水稻为试材,研究盆栽及田间条件下转Bt基因及亲本水稻苗期、拔节期、抽穗期和成熟期倒1叶光合特性及其相关光合酶活性、光合产物积累的动态变化。【结果】转Bt基因及亲本水稻生理活动峰值均出现在拔节期。与亲本水稻相比,转Bt基因水稻苗期叶片净光合速率显著低于亲本;而拔节期和抽穗期,转Bt基因水稻倒1叶净光合速率、叶绿素含量和乙醇酸氧化酶活性显著高于亲本水稻（P＜0.05）,且拔节期转Bt水稻倒1叶气孔导度和胞间CO2浓度也显著高于亲本水稻（P＜0.05）;成熟期,转Bt基因与亲本水稻倒1叶光合特性无显著性差异（P＞0.05）。【结论】与亲本水稻相比,外源Bt基因的插入对水稻叶片光合特性产生短暂影响,但这种影响不具有持续性。