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为了了解嗜线虫致病杆菌Xenorhabdus nematophila几丁质酶的功能,本研究通过PCR技术从X.nematophila HB310菌株中克隆得到了几丁质酶基因chi70,对该基因进行了生物信息学分析。结果表明该基因全长1947 bp,编码648个氨基酸,表达的Chi70蛋白不含信号肽。将chi70基因进行原核表达,获得大量重组Chi70蛋白。采用二硝基水杨酸法(DNS法)测定重组蛋白酶活,结果表明几丁质酶Chi70在pH7.0时活性最高,为8.815 U/mL。采用生测方法测定Chi70对苏云金芽孢杆菌HD-73(Bt HD-73)的增效活性,结果表明单独使用Bt HD-73时对2龄棉铃虫幼虫的LC50为14.80μg/m L,添加Chi70后的Bt HD-73对2龄棉铃虫幼虫的LC50为5.66μg/m L,实验结果说明Chi70对Bt具有明显的增效作用。
In order to understand the function of Xenorhabdus nematophila chitinase, the chitinase gene chi70 was cloned from X. nematophila HB310 by PCR and the bioinformatics analysis was carried out. The results showed that the gene was 1947 bp in length and encoded 648 amino acids. The expressed Chi70 protein contained no signal peptide. The chi70 gene prokaryotic expression, access to a large number of recombinant Chi70 protein. The recombinant protease activity was measured by the dinitrosalicylic acid method (DNS method). The results showed that chitinase Chi70 had the highest activity at pH 7.0 with 8.815 U / mL. The synergistic activity of Chi70 on Bacillus thuringiensis HD-73 (Bt HD-73) was determined by bioassay. The results showed that the LC50 value of larvae of H. armigera larvae was 14.80μg / m L when Bt HD-73 was used alone, The LC50 of the Bt HD-73 to the 2nd-instar larvae of H. armigera was 5.66 μg / mL. The experimental results showed that Chi70 had a synergistic effect on Bt.