Chen Jian-Wu, Tang Li-Xia, Song Shao-Yun, Yuan Mei-Jin, Pang Yi
State Key Laboratory for Biocontrol and Institute of Entomology, Zhongshan University, Guangzhou 510275, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):538-44.
Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild-type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD-1 were found to contain the vip3A genes. Because acrystallerous strain Cry- B derived from Btk HD-1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild-type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM-T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A (a), Vip3A(b), Vip3A-S, Vip3A-S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP-S101 and pOTP-611 were constructed by in- serting 2 vip3A genes (vip3A-S101 and vip3A-611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein. Experiments showed that the level of soluble proteins of Vip3A-S101 in E. coli M15[pOTP-S101] and Vip3A-611 in E. coli M15 [pOTP-611] were about 48% and 35% respectively. Bioassay showed that each of these Vip3A proteins had similar toxicity against neonate Spodoptera litura larvae, indicating that some amino acids change had little effect on the insecticidal activity of proteins. Although vip3A genes are conservative, the unknown insecticidal spectrum is still to be brought out. Vip3A genes can be used for the construction of the Bt engineered strains and transgenic plants. In addition, vip3A genes are excellent candidates for delay of the pest resistance due to the difference of the action model from that of Bt delta-endotoxins.
Vip3A是一种新型杀虫蛋白，由苏云金芽孢杆菌（Bt）在营养生长阶段分泌。Vip3A蛋白对多种鳞翅目昆虫幼虫具有杀虫活性。自首次从Bt中克隆出vip3A基因以来，又分离出了许多其他的vip3A基因。为了研究vip3A基因对Bt的贡献并反映其进化关系，对含有vip3A基因的菌株进行了筛选并分析了基因相似性。使用PCR扩增技术，从不同地区分离得到的114株野生型苏云金芽孢杆菌（Bt）菌株以及巴斯德研究所的41株标准Bt菌株中筛选vip3A基因。发现包括苏云金芽孢杆菌库斯塔克亚种（Btk）HD-1在内的39株菌株含有vip3A基因。由于源自Btk HD-1的无晶体菌株Cry-B被证明不含有vip3A基因，推测vip3A基因可能位于质粒上。用多克隆抗体通过蛋白质免疫印迹法检测这些菌株中表达的Vip3A蛋白，其中有4株未表达Vip3A蛋白。从对鳞翅目昆虫幼虫具有不同活性水平的野生型苏云金芽孢杆菌菌株S101和611中扩增出的vip3A基因被克隆到pGEM-T Easy载体中。将这2种假定的Vip3A蛋白与GenBank数据库中的其他6种（Vip3A (a)、Vip3A(b)、Vip3A-S、Vip3A-S184、Vip83和Vip3V）以及2种已报道的Vip3A蛋白（Vip14和Vip15）进行比对，结果表明vip3A基因具有高度保守性。通过将2个vip3A基因（vip3A-S101和vip3A-611）分别插入表达载体pQE30中构建了质粒pOTP-S101和pOTP-611，并将其转化到大肠杆菌M15中。用1 mmol/L IPTG诱导携带pOTP质粒的大肠杆菌M15细胞表达89 kDa的蛋白。实验表明，大肠杆菌M15[pOTP-S101]中Vip3A-S101的可溶性蛋白水平约为48%，大肠杆菌M15 [pOTP-611]中Vip3A-6
