对粉纹夜蛾高毒力cry9Ea基因的克隆及表达

【目的】从本实验室分离的Bt4菌株中克隆cry9Ea基因,并研究其表达和杀虫活性。【方法】以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因。【结果】将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcry9Ea。转化E.coli BL21(DE3),诱导后表达130kDa的蛋白,再将cry9Ea7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定。生物活性测定结果显示Cry9Ea7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC50为0.044μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性。【结论】克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Ea7,并成功构建了工程菌BioHD9Ea7。     

对粉纹夜蛾高毒力cry9Eα基因的克隆及表达

[目的]从本实验室分离的Bt4菌株中克隆cry9Eα基因,并研究其表达和杀虫活性.[方法]以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因.[结果]将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcrygEa.转化E.coli BL21(DE3),诱导后表达130 kDa的蛋白,再将cry9Eα7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定.生物活性测定结果显示CrygEa7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC_(50)为0.044 μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性.[结论]克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Eα7,并成功构建了工程菌BioHD9Ea7.     

转cry1Ab/cry1Ac基因玉米Cry1Ab/Cry1Ac融合蛋白表达及对亚洲玉米螟的室内杀虫效果

【目的】室内抗螟性评价是转Bt基因抗虫玉米研发和安全性评价的重要环节。【方法】采用酶联免疫吸附测定法(ELISA)测定了转cry1Ab/cry1Ac基因玉米ZZM030心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量;采用室内生测法测定了分别取食转基因玉米ZZM030和非转基因玉米X249心叶后亚洲玉米螟Ostrinia furnacalis敏感品系ACB-BtS、Cry1Ab抗性品系ACB-AbR和Cry1Ac抗性品系ACB-AcR初孵幼虫的存活率。【结果】转基因抗虫玉米ZZM030 4叶期和8叶期心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量分别是10.62和2.94 μg/g FW。敏感品系亚洲玉米螟初孵幼虫取食转基因玉米ZZM030心叶2 d的存活率仅为23.6%,4 d后存活率为0,而取食非转基因对照玉米X249心叶4 d的存活率高达93.1%。Cry1Ab抗性品系和Cry1Ac抗性品系初孵幼虫取食转基因玉米ZZM030心叶6 d后的存活率分别为11.1%和12.5%,而取食非转基因玉米X249心叶6 d后的存活率分别为81.9%和77.8%。【结论】转cry1Ab/cry1Ac基因玉米ZZM030心叶中高表达的Cry1Ab/Cry1Ac融合蛋白对亚洲玉米螟初孵幼虫具有极高的杀虫效果。     

苏云金芽孢杆菌沉默基因cry2Ab3在大肠杆菌中表达及杀虫活性研究

对苏云金芽孢杆菌C002菌株cry2Ab基因阳性克隆pHT3152Ab进行亚克隆和序列测定,在CenBank注册后经国际Bt杀虫蛋白基因委员会正式命名为cry2Ab3。序列分析表明该基因含有芽孢杆菌特异的RBS序列,但没有功能性启动子,为沉默基因。根据大肠杆菌T7表达载体pET21b克隆位点和cry2Ab3开放阅读框架(ORF)两端序列,设计合成一对特异引物L2ab5和L2ab3,高保真PCR扩增获得cry2Ab3完整ORF,经酶切、连接构建了重组表达质粒pET2Ab3。表达质粒导入大肠杆菌BL21(DE3),IPTG诱导后,SDSPAGE电泳证实了cry2Ab3的表达。生物测定显示诱导培养物对棉铃虫初孵幼虫和小菜蛾二龄幼虫具有杀虫活性,能明显抑制二化螟二龄幼虫生长,但对甜菜夜蛾和玉米螟没有明显活性。进一步提取Cry2Ab3蛋白,生测结果表明其对棉铃虫LC50为32.55μg/g。     

Bt菌株QCL-1中cry2Ac10基因的克隆、表达和活性研究

目的:从高毒力Bt菌株中克隆cry2Ac10基因,并研究其表达和杀虫活性。方法:以Bt菌株QCL-1质粒为模板,利用cry2特异性引物FY2A5和FY2A3进行PCR扩增,将目的片段克隆到表达载体pET-21b( ),构建T7启动子控制的大肠杆菌重组表达质粒pET21b-cry2Ac。经IPTG诱导后,SDS-PAGE检测基因表达情况,然后对表达产物进行生物活性测定。结果:从菌株QCL-1中克隆出目的基因,该基因的编码框由1 872个碱基组成,编码的蛋白质由623个氨基酸组成,与已报道的Cry2Ac氨基酸同源性为97.4%~99.7%。该基因(GenBank accession EF405952)已被国际Bt基因命名委员会正式命名为cry2Ac10。该基因在大肠杆菌BL21(DE3)中能够正常表达70kDa的蛋白,表达产物对棉铃虫、粘虫和粉纹夜蛾幼虫具有高毒力,同时对甜菜夜蛾幼虫生长有抑制作用,其中对棉铃虫和粘虫初孵幼虫的LC50分别为30.0μg/g和16.7μg/g。结论:成功克隆和表达了cry2Ac10基因,并明确了cry2Ac10蛋白的活性,为该基因的研究和应用奠定基础。     

苏云金芽孢杆菌B-Pr-88菌株中cry2Ab4基因的表达和杀虫活性研究

以我室自行分离的对鳞翅目夜蛾科害虫具有高毒力的Bt菌株B-Pr-88为材料,用PCR-RFLP方法从其质粒DNA文库中筛选到含cry2Ab基因的一个阳性克隆pZF858,序列测定发现,该片段含有cry2Ab全长基因,开放读码框为1902bps,编码由633个氨基酸组成的70.7kD蛋白,氨基酸同源性与已公布的cry2Ab基因同源性均为99.8%,经Bt基因国际命名委员会正式命名为cry2Ab4。根据cry2Ab4基因开放阅读框架(ORF)两端序列,设计合成一对特异引物L2ab5和L2ab3,PCR扩增获得cry2Ab4完整ORF,与大肠杆菌表达载体pET-21b连接,构建了重组表达质粒pET-2Ab4,质粒导入大肠杆菌BL21(DE3),IPTG诱导后,SDS-PAGE电泳证实该基因表达了60kD的蛋白,生物测定表明,Cry2Ab4对棉铃虫和大豆食心虫具有高毒力,同时对小菜蛾和二化螟有一定的杀虫活性,而对亚洲玉米螟和甜菜夜蛾没有杀虫活性。     

苏云金芽孢杆菌cry1Ab13基因的克隆及表达研究

BtC0 0 5是我国自行分离的对多种害虫具有毒杀作用的苏云金芽孢杆菌 ,经PCR RFLP系统鉴定 ,它含有cry1Ab基因。Southernblot结果显示 :PstI酶切C0 0 5质粒所得的 8 5kb长的DNA片段为cry1Ab基因的阳性杂交带。以pUCP1 9为载体 ,克隆了该片段并证明其含有cry1Ab基因。对其进行亚克隆和测序 ,结果表明该基因编码区为 3 4 6 8bp ,其编码的蛋白含1 1 5 5个氨基酸 ,分子量为 1 3 0 6kD ,等电点为pH4 845。该基因已在GenBank基因库中注册 ,Accessionnumber为AF2 5 4 6 4 0 ,并为国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Ab1 3。将cry1Ab1 3基因在Bt无晶体突变株cryB- 中表达 ,蛋白质电泳结果表明在 1 3 0kD处有表达带 ,并证明CryAb对小菜蛾有较高的杀虫活性。     

31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明：25株含cry1基因,表达蛋白130～150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明：cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。     

苏云金芽胞杆菌cry2Ad基因的克隆及其表达产物的活性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)SBT2是我国新分离出的一株野生菌株.扫描电镜显示该菌株产生双锥体形晶体.琼脂糖凝胶电泳发现其质粒图谱含有5个条带.聚丙烯酰胺凝胶电泳显示此菌株产生130 kD晶体蛋白.利用PCR-RFLP法进行杀虫基因类型鉴定,发现其含有cry1Aa、cry1Da、cry1Hb、cry1Jb、cry1Ka 、cry1Ib、基因.Cry2Ad蛋白的活性至今未见研究报道,本研究克隆和测序了该基因.并对其进行了表达.生物活性测定结果表明其表达产物对舞毒蛾(Lymantria dispar)、棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)、小菜蛾(Plutella xylostella)有低活性;对大猿叶甲(Colaphellus bowringi)无活性.     

苏云金芽胞杆菌V4菌株cry杀虫基因的鉴定、表达及杀虫活性分析

旨在给生物防治提供更多的基因资源,以从本实验室分离的300株苏云金芽胞杆菌基因组为模板,利用cry1类全长通用引物进行PCR cry1类基因鉴定。经过PCR-RFLP鉴定出菌株V4含有cry1Ea基因,进一步研究发现该菌株还含有cry2Aa基因,并成功克隆到了这两个新基因,被Bt国际命名委员会正式命名为cry1Ea12和cry2Aa16。将两种基因在大肠杆菌Rosetta(DE3)中表达,表达蛋白进行杀虫活性测定,结果表明两种蛋白杀虫活性不理想,但对试虫存在明显的体重抑制,而V4菌株蛋白具有较高的杀虫活性。     

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.     

cry4Aa、cry4Ba和cry11Aa基因在苏云金杆菌无晶体型菌株中的表达

利用穿梭载体pBU4,将苏云金杆菌以色列亚种（Bti)的cry4Aa、cry4Ba和cry11Aa基因分别转入Bti无晶体突变株4Q7中,获得了转化菌株Bt-B601、Bt-B611和Bt-B640。SDS－PAGE结果显示：cry4Aa、cry4Ba和cry11Aa蛋白均分别获得了表达。透射电镜下观察,转化菌 有产生球形或菱形伴胞晶体。转化菌株对敏感和抗性致倦库蚊及白纹伊蚊幼虫的生物测定结果显示：cry4Aa、cry4Ba和cry11Aa蛋白对库蚊和伊蚊的毒力较低,二元毒素抗性库蚊幼虫对Bti杀蚊毒素蛋白无明显的交叉抗性。     

Cryptochrome photoreceptors cry1 and cry2 antagonistically regulate primary root elongation in Arabidopsis thaliana

Cryptochromes are blue-light receptors controlling multiple aspects of plant growth and development. They are flavoproteins with significant homology to photolyases, but instead of repairing DNA they function by transducing blue light energy into a signal that can be recognized by the cellular signaling machinery. Here we report the effect of cry1 and cry2 blue light receptors on primary root growth in Arabidopsis thaliana seedlings, through analysis of both cryptochrome-mutant and cryptochrome-overexpressing lines. Cry1 mutant seedlings show reduced root elongation in blue light while overexpressing seedlings show significantly increased elongation as compared to wild type controls. By contrast, the cry2 mutation has the opposite effect on root elongation growth as does cry1, demonstrating that cry1 and cry2 act antagonistically in this response pathway. The site of cryptochrome signal perception is within the shoot, and the inhibitor of auxin transport, 1-N-naphthylphthalamic acid, abolishes the differential effect of cryptochromes on root growth, suggesting the blue-light signal is transmitted from the shoot to the root by a mechanism that involves auxin. Primary root elongation in blue light may thereby involve interaction between cryptochrome and auxin signaling pathways.     

RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from India

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.     

cry4Aa、cry4Ba和cryllAa基因在苏云金杆菌无晶体型菌株中的表达

利用穿梭载体pBU4,将苏云金杆菌以色列亚种(Bti)的cry4Aa、cry4Ba和cry11Aa基因分别转入Bti无晶体突变株4Q7中,获得了转化菌株Bt-B601、Bt-B611和Bt-B640.SDS-PAGE结果显示Cry4Aa、Cry4Ba和Cry11Aa蛋白均分别获得了表达.透射电镜下观察,转化菌株能产生球形或菱形伴胞晶体.转化菌株对敏感和抗性致倦库蚊及白纹伊蚊幼虫的生物测定结果显示Cry4Aa、Cry4Ba和Cry11Aa蛋白对库蚊和伊蚊的毒力较低,二元毒素抗性库蚊幼虫对Bti杀蚊毒素蛋白无明显的交叉抗性.     

转cry1Ac+cry2Ab基因棉与转cry1Ac+EPSPS基因棉荒地的生存竞争能力

【背景】此研究为"十二五"转基因生物新品种培育国家项目中创建新的转基因棉花品种环境安全评价技术而设。【方法】以转双价双Bt抗虫基因(cry1Ac+cry2Ab)棉和转双价抗虫、抗除草剂基因(cry1Ac+EPSPS)棉为观察品种,非转基因棉赣棉11号为对照品种,在荒地用撒播和3cm深度播种2种方式,于2011年5月～2012年3月对棉花出苗率、株高、生育进程、棉吐絮瓣数、絮瓣脱落率、自生苗等生存竞争能力进行比较,检测、评价其杂草化的风险,并探讨、验证检测技术的可行性。【结果】在荒地条件下,以2种方式播种的转cry1Ac+cry2Ab基因棉和转cry1Ac+EPSPS基因棉与非转基因棉相比,上述各项指标的竞争能力总体上未表现显著优势。【结论与意义】转cry1Ac+cry2Ab基因棉和转cry1Ac+EPSPS基因棉在荒地条件下生长无杂草化风险。同时,研究证明,在荒地自然生态条件下,可以采用撒播和3cm深度播种方法检测新的转基因棉花品种在生存竞争能力上的杂草化风险,在测评上有互为参照效应,为定性评价新的转基因棉花品种的杂草化风险提供了保障。     

Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus.

Genes cry26Aal and cry28Aal were cloned from Bacillus thuringiensis ssp. finitimus strain B-1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aal gene product included seven residues determined to be an N-terminal part of a chymotrypsin-treated delta-endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore-associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aal in both types of crystal bodies is synthesized under the control of one and the same genomic locus.     

Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain T03B001

Three cry9 genes, cry9Da4, cry9Eb2, and cry9Ee1, were cloned from Bacillus thuringiensis strain T03B001 using a high-resolution melting analysis method. All three cry9 genes were overexpressed in Escherichia coli Rosetta (DE3), and the expressed products Cry9Eb2 and Cry9Ee1 were shown to be toxic to Plutella xylostella and Ostrinia furnacalis, but not to Helicoverpa armigera or Colaphellus bowringi. The bioassay of Cry9Eb2 and Cry9Ee1 against Cry1Ac-resistant P. xylostella strains indicated that both novel Cry9 toxins exhibited no cross-resistance with Cry1Ac. Cry9Eb2 and Cry9Ee1 can be applied not only for P. xylostella and O. furnacalis control, but also for the Cry1Ac-resistance management of pests.