利用荧光指示分子IeGFP比较vip3A和cry1Ia基因启动子活性

苏云金芽孢杆菌（Bt）的Vip3A和Cry1Ia蛋白质序列无同源性,但具有其他相似的特征,例如在孢子形成早期合成,然后跨膜转运至胞外。本研究以新型融合荧光蛋白IeGFP为报告分子,比较vip3A（Pv）和cry1Ia基因（Pi）启动子的调控模式和活性。结果表明,在大肠杆菌中,两者均为组成型启动子。通过荧光信号强度的比较发现,在大肠杆菌中,Pi活性显著强于乳糖操纵子调节基因启动子（PlacI, P<0.01）,但比PlacI增强型突变体（PlacIq, P<0.01）和cry1Ac基因启动子（Pac, P<0.01）弱。在3个Bt启动子中,Pv活性最弱,接菌12 h后,与PlacI接近（P>0.05）。在Bt菌株中,Pv对IeGFP的表达调控与之前报道的Pi相似。启动子的Shine-Dalgarno（SD）序列与目的基因起始密码子（AUG）之间的间隔区域在调节细菌的蛋白质翻译效率方面发挥重要作用。本研究中构建的大部分Pv与Iegfp基因的间隔区域突变,均导致相应大肠杆菌和Bt细胞的荧光强度显著降低（P<0.05）,说明融合荧光蛋白IeGFP具有较好的灵敏度。该研究不仅确定了2种启动子在Bt和大肠杆菌中的活性,而且验证了IeGFP指示弱启动子活性的可行性。     

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa + pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.     

土壤中对甲虫有活性苏云金芽胞杆菌的筛选

近几年来,甲虫已成为十字花科蔬菜的头号害虫。本文采集了湖北省各地区192份土壤样品,分离到苏云金芽胞杆菌(简称Bt)菌株74株,以甲虫代表——黄粉虫为靶标昆虫筛选了15株典型Bt,得到一株对甲虫有活性的Bt L1;生物测定该菌的半致死浓度(LC50)为221.20μg·g-1;聚丙烯酰胺凝胶电泳(SDS-PAGE)显示L1至少具有3个杀虫晶体蛋白;PCR扩增杀虫晶体蛋白基因,测序发现Bt L1中含有cry1Ac,cry1Aa,cry1Ia基因,推测Bt L1中对甲虫活性的杀虫晶体蛋白基因为cry1Ia。     

Mosquito larvicidal activity of transgenic Anabaena strain PCC 7120 expressing combinations of genes from Bacillus thuringiensis subsp. israelensis.

Various combinations of the genes cryIVA (cry4A), cryIVD (cry11A), and p20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 by means of Escherichia coli-Anabaena shuttle vector pRL488p and were expressed under control of two tandem strong promoters, a cyanobacterial promoter (PpsbA) and an E. coli T7 promoter (PA1). Two of the clones carrying cryIVA plus cryIVD, one with p20 and one without p20, displayed toxicity against third-instar larvae of Aedes aegypti at levels greater than any level previously reported for transgenic cyanobacteria.