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为研究苏云金芽胞杆菌杀虫晶体蛋白在细胞中的定位及在细胞中的形成,构建了Cry1Ac-GFP融合蛋白,大小约为160kD.将携带cry1Ac启动子的cry1Ac-gfp融合基因片段克隆到pHT304载体上,获得融合表达载体pHTcry1Ac-gfp.pHTcry1Ac-gfp转化到无晶体突变株HD-73cry-中,获得融合表达菌株HD-73-(pHTcry1Ac-gfp).gfp基因通过同源重组插入到HD-73内源大质粒pHT73上cry1Ac基因的3′端,获得原位融合表达菌株HD-73Φ(cry1Ac-gfp)3534.激光共聚焦显微镜和Western杂交分析表明,不对称隔膜形成时,HD-73-(pHTcry1Ac-gfp)和HD-73Φ(cry1Ac-gfp)3534细胞中检测到Cry1Ac-GFP融合蛋白的表达.融合蛋白颗粒在细胞中的聚集存在一定的极性,分布于母细胞不对称隔膜附近.Cry1Ac-GFP和Cry1Ac蛋白对小菜蛾的杀虫活性在95%置信区间内没有明显差异.
In order to study the localization of the insecticidal crystal protein of Bacillus thuringiensis in cells and its formation in cells, a Cry1Ac-GFP fusion protein was constructed with a size of about 160 kD.The cry1Ac-gfp fusion gene fragment carrying cry1Ac promoter was cloned into pHT304 vector (PHTcry1Ac-gfp). The gfp gene was inserted into HD-73 by homologous recombination, and then transformed into HD-73cry-, the fusion expression vector pHTcry1Ac-gfp.pHTcry1Ac- The in situ fusion expression strain HD-73Φ (cry1Ac-gfp) 3534 was obtained at the 3 ’end of the cry1Ac gene of the endogenous plasmid pHT73. Confocal laser scanning microscopy and Western blotting showed that HD-73- ( Cry1Ac-GFP fusion protein was detected in pHTcry1Ac-gfp and HD-73Φ (cry1Ac-gfp) 3534. The fusion protein particles were aggregated in the cells with a certain polarity and distributed in the vicinity of the asymmetric septum of blast cells.Cry1Ac Insecticidal activity of -GFP and Cry1Ac proteins against Plutella xylostella was not significantly different at 95% confidence intervals.