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[目的]获得aveD基因定点突变株,为阿维菌素的遗传育种提供理论依据。[方法]采用重叠延伸PCR技术对aveD基因进行定点突变,并通过体外酶切连接等分子生物学操作,构建了aveD基因的定点突变载体pDC3(pKC1139∷aveD、),导入阿维菌素(Avermectin)产生菌阿维链霉菌(Streptomyces avermitilis)76-9的aveD基因缺失突变株489中。[结果]经过同源重组,获得aveD基因定点缺失突变株536。测序结果表明突变株536,aveD基因编码区中第69位碱基C突变为T,相应的氨基酸序列第23位由Thr突变为Ile。[结论]该研究为研制生产阿维菌素B的基因工程菌奠定了基础。
[Objective] The aim of this study was to obtain the mutated point aveD gene and to provide a theoretical basis for the genetic breeding of avermectin. [Method] The aveD gene was mutated by overlap extension PCR and the aveD site-directed mutagenesis vector pDC3 (pKC1139 ::aveD,) was constructed by in vitro enzymatic ligation and other molecular biology procedures. Avermectin ) Produced the aveD gene deletion mutant 489 of Streptomyces avermitilis 76-9. [Result] After homologous recombination, the aveD gene deletion mutant 536 was obtained. Sequencing results showed that the mutation of 536 in the coding region of aveD gene in mutant 536 was T and the 23rd in the corresponding amino acid sequence was changed from Thr to Ile. [Conclusion] This study laid the foundation for the development of gene engineering bacteria producing abamectin B.