Cry1A型杀虫晶体蛋白活性区的空间结构比较分析

对9种代表性Cry1A型杀虫晶体蛋白成员进行活性区空间结构的同源模建与比较分析.分析结果表明:Domain Ⅰ和Domain Ⅲ相对保守,其中,Domain Ⅰ整体走向及结构基本重叠,只有Cry1Aa和Cry1Af多了一个螺旋;Domain Ⅱ的主要差异体现在loop上;将Domain Ⅲ结构一致的成员Cry1Ab,Cry1Ad,Cry1Ae和Cry1Af归为一个亚型,其他5种成员归为另一个亚型.研究确定了影响Cry1A型杀虫晶体蛋白结构差异的关键氨基酸及关键结构片段.     

苏云金芽孢杆菌毒素Cry7Aa1三维结构模型

运用生物信息学软件和网络数据库资源,采取同源建模的方法以毒素蛋白Cry8Ea1为模板预测了苏云金芽孢杆菌毒素Cry7Aa1的三级结构并分析了相关的生物学功能.结果表明,Cry7Aa1为3个结构域组成的近似球状蛋白,Cry7Aa1在氨基酸序列上与Cry8Ea1有40%的同源性,在三维结构_ECry7Aa1与Cry8Ea1有高度的相似性,它们结构域Ⅰ几乎完全相同,在结构域Ⅱ之间和结构域Ⅲ之间存在较小差异.Cry7Aa1与Cry8Ea1的表面电势分布不同.2种毒素具有相似的毒杀机理.模型经Ramachandran图和Verify 3D检验具有合理性.     

小菜蛾钙黏蛋白PxCR_(10-11)结构域的克隆、原核表达及功能分析

钙黏蛋白(cadherin)是一类跨膜糖蛋白,因其在苏云金芽胞杆菌(Bacillus thuringiensis,Bt)的杀虫过程中作为主要的受体而受到广泛研究.钙黏蛋白的特征之一是由若干钙黏蛋白重复单元(cadherin repeats,CR)组成的长链状蛋白,其中靠近细胞膜的CR结构域被认为是钙黏蛋白与Bt毒素发生互作的区域.小菜蛾(Plutella xylostella)钙黏蛋白由11个CR结构域和1个跨膜结构域组成,其中第10和11个CR结构域PxCR_(10-11)被认为是钙黏蛋白与Bt毒素的互作区域.本研究从小菜蛾中肠cDNA中克隆得到小菜蛾钙黏蛋白PxCR_(10-11)结构域的DNA片段,并通过原核表达系统对PxCR_(10-11)结构域进行表达.配体印迹结果表明PxCR_(10-11)可以特异性和Cry2Ab结合;杀虫实验结果表明PxCR_(10-11)能提高Cry2Ab对小菜蛾的毒力.此外,利用蛋白质同源建模和糖基化位点预测网站对PxCR_(10-11)蛋白质结构进行了分析.上述结果表明PxCR_(10-11)可能参与了Cry2Ab毒素对小菜蛾的毒杀过程,为后续研究小菜蛾钙黏蛋白和Cry2Ab的相互作用机制奠定了基础.     

聚合Cry1Ab和Cry1F基因抗虫玉米对亚洲玉米螟存活率及平均体重的影响

实验用聚合Cry1Ab和Cry1F基因抗虫玉米335YH、696YH的人工饲料饲养亚洲玉米螟幼虫,研究人工饲料中聚合基因玉米对其存活率和平均体重的影响.结果表明:在25%用量处理下,玉米螟幼虫的存活率均低于38%,平均体重均低于45 mg,说明聚合Cry1Ab和Cry1F基因抗虫玉米种子能抑制供试亚洲玉米螟幼虫的生长和存活.335YH 25%用量和50%用量的致死性差异不大,但50%用量比25%用量抑制幼虫生长的效果更好.696YH品种对幼虫的致死性和抑制生长效果受剂量影响较大.     

含Cry1Ab/Ac基因的白僵菌工程菌的构建

以来源于苏云金芽孢杆菌的Bt-Cry1Ab/Ac基因为目的基因,以潮霉素Hyg为抗性标记基因,以绿色荧光蛋白基因GFP为报告基因的真菌表达载体,采用农杆菌介导真菌遗传转化(ATMT)法,将Cry1Ab/Ac基因插入白僵菌基因组中,先后用潮霉素B抗性筛选、荧光检测、真菌基因组PCR分子检测法、Bt-Cry1Ab/Ac基因检测试纸条多种方法进行转基因工程菌株鉴定,结果证实了目的基因成功插入白僵菌基因组,为白僵菌杀虫活性的深入研究奠定了基础.     

尼古丁生物降解产物中烟碱型乙酰胆碱受体α7亚型靶向化合物的虚拟筛选

在以微生物降解尼古丁的代谢途径中,已被分离出多种与尼古丁结构类似的中间产物.烟碱型乙酰胆碱受体α7亚型(α7-n AChR)是阿尔茨海默病和多种炎症药物研发的重要靶点,而尼古丁是与其特异性结合的天然配体.计算机虚拟筛选技术是现代新药研发的一种重要方法.采用尼古丁降解产物及其结构相似物与α7-n AChR进行活性位点对接及分子计算,探寻是否可以从尼古丁的降解产物中寻找出新型以α7-n AChR为靶点的小分子治疗药物.选用9个尼古丁代谢的中间产物和与其结构相似的78个化合物为筛选对象.计算结果显示:9个尼古丁代谢中间产物与α7-n AChR的结合能量在-5.7 kcal/mol~-6.7 kcal/mol之间,3个结构相似化合物与α7-n AChR的结合能量约为-8.0 kcal/mol.这些化合物均可以与α7-n AChR的活性区域进行结合,其结合能与其天然配体接近或更好.筛选的结果为此类化合物的下一步的药理学研究提供了参考.     

苏云金芽孢杆菌cyt1Aa基因在拟步甲亚种菌株中的表达及重组菌株杀虫活性研究

将含苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis,简称Bti)cyt1Aa基因的质粒电激转化至对鞘翅目害虫有专一活性的Bt拟步甲亚种(Bacillus thuringiensis subsp.tenebrionis,简称Btt)菌株中进行表达,获得重组菌株Btt-WF45。对表达产物进行SDS-PAGE分析,显示Cyt1Aa蛋白获得了大量表达。生物测定结果表明,Btt-WF45对鞘翅目小圆叶甲(Plagiodera Redtenbacher)幼虫没有毒力,对致倦库蚊(Culex quinquefasciatus)的毒力比对照菌株4Q7-WF45高0.33倍,对白纹伊蚊(Aedes albopictus)的毒力比4Q7-WF45高0.63倍。由于在重组菌株Btt-WF45中Cry3A蛋白的表达量太低,所以在该结果中未能显示Cyt1Aa蛋白与Cry3A蛋白是否存在协同增效作用。     

农杆菌介导的Bt基因转化马铃薯的研究

马铃薯块茎蛾的危害极为严重,通过植物基因工程将Bt基因的Cry1Ab类型导入马铃薯,以期获得抗虫性提高的马铃薯基因工程植株.以马铃薯品种‘会-2’为受体材料,对影响转化效率的因素进行了优化,结果表明:3号(1/2MSO+50μLAs+2 mL农杆菌悬浮液)浸染液有利于抗性芽的分化,乙酰丁香酮可以提高叶片的转化率,共培养3 d的分化率较高.共获得经过卡那霉素筛选的114个转化植株,对初筛株系进行PCR分子鉴定,结果有55%的植株扩增出目的条带,初步证明Cry1Ab基因已整合到马铃薯的基因组中.转化植株的抗虫性鉴定正在进行中.     

吉兰-巴雷综合征患者外周血清白介素2、干扰素α及抗体水平的检测

目的探讨白介素2（IL-2）、抗白介素2抗体（IL-2Ab）、干扰素口（IFN-α）和抗干扰素α抗体（IFN-αAb）在吉兰-巴雷综合征（GBS）患者病理免疫机制中的作用和意义．方法采用ELISA检测了50例GBS病人和30名健康人血清IL-2、IL-2Ab、IFN-α和IFN—αAb，并进行比较．结果GBS患者血清IL-2、IL-2Ab、IFN-α和IFN—αAb均比正常对照组明显升高．结论IL-2、IFN-α及其相应抗体可能参与了GBS的免疫病理过程，IL-2Ab和IFN-αAb可能作为中和性抗体对IL-2和IFN-α起负调节作用，且可能与疾病的自限有关．     

抗Cry2Ad毒素小菜蛾抗菌肽的分离、纯化及特性研究

通过比较饲喂Cry2Ad诱导的抗Cry2Ad毒素品系和敏感品系小菜蛾抗菌肽的含量和抑菌活性,结果显示,抗Cry2Ad小菜蛾被喂食Cry2Ad毒素后,体内抗菌肽含量显著增高,抑菌活性更强。说明小菜蛾抗菌肽与小菜蛾对Cry2Ad毒素的抗性有一定的相关性。通过SDS-PAGE电泳检验抗菌肽分子量,得到抗Cry2Ad毒素小菜蛾抗菌肽有一分子量约为7.8 KDa特别条带,采用截留量10 KDa的超滤管和阴、阳离子交换柱层析对抗Cry2Ad小菜蛾抗菌肽进行分离、纯化,得到具有抑菌活性的抗菌肽。     

The role of β18-β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

The β18-β19 loop in domain Ⅲ of Cry1Ac toxin is unique among Bacillus thuringlensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 revealed that a charged amino acid in this position would cause very unfavorable influence on insecticidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin.     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

桃蚜关键抗性基因挖掘及抗蚜Cry蛋白预测

为了挖掘桃蚜(Myzus persicae)的关键抗性基因并构建抗性调控网络,通过加权基因共表达网络分析(WGCNA)和差异表达基因分析(DEGs)对桃蚜抗性研究的GEO数据库进行分析,筛选出2 426个枢纽基因和2 263个差异表达基因探针.将关键抗性基因在String数据库中进行蛋白质相互作用(PPI)分析,获得154个桃蚜关键抗性基因,并绘制桃蚜的抗性调控网络.结果表明:acpp基因的上调表达可能是大多数Cry蛋白不能有效杀死桃蚜的原因之一;参考抗蚜Cry1Cb2蛋白和11个靶标蛋白的对接规则,通过同源建模和分子对接等生物信息学技术,预测了10个新的Cry蛋白可能对桃蚜具有杀虫活性.     

Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development ofHelicoverpa armigera

The resistance ratio ofHelicoverpa armigera to Cry1 Ac insecticidal protein fromBacillus thuringiensis (Bt) is 13.1- and 3.02-fold after 18 generations of selection by transgenic tobacco expressing Bt or two (Bt and CpTI) insecticidal protein genes, in which the average corrected mortality for each selection treatments is about 60%. The mortality of selected population by transgenic Bt gene tobacco is significantly lower than the control strain when fed on transgenic tobacco plants. The mortaltty of the selected population by transgenic two genes tobacco was not significantly different from the control strain. This is the first experiment under laboratory condition which has proved that transgenic two genes tobacco could significantly delay resistance development ofH. armigera compared with one gene.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

苏云金芽孢杆菌猝倒亚种Cry1Aa13基因的克隆及序列分析

根据Gen Bank中Cry1A类基因序列设计一对特异性引物，以苏云金芽孢杆茵猝倒亚种质粒DNA为模板，应用PCR扩增技术得到一大小约为3．6kb的DNA片段(Cry1Aa13)．通过引物步行法测定该片段长3598bp，其中开放阅读框(ORF)长3540bp，编码1180个氨基酸，分子量为133．49kD，等电点pI=5．0．序列比较表明该基因与Cry1Aa类基因高度同源(达95％以上)，该基因已在GenBank中登录，登录号为AF510713，并被Btδ-内毒素基因国际命名委员会正式命名为Cry1Aa13。