芒果炭疽病菌小柱孢酮脱水酶基因SCD1的克隆与敲除载体构建

Cloning and Construction of Knockout Vector of Scytalone Dehydratase Gene SCD1 from Mango Anthracnose(Colletotrichum gloeosporioides)

芒果炭疽病(Colletotrichum gloeosporioides)是为害芒果的重要病害之一,为了明确小柱孢酮脱水酶基因SCD1与芒果炭疽病菌致病力之间的相互关系,本研究以芒果炭疽病菌DNA为模板,利用同源克隆技术扩增SCD1,分析其序列特征、推测了其蛋白的保守结构域,并借助In-Fusion~ HD Cloning Kit技术进行敲除载体构建。结果表明,该基因DNA和cDNA全长分别为796 bp、564 bp,编码区有两个内含子(大小为52 bp,180 bp),推测编码187个氨基酸,其分子量约为21.52 kD,等电点PI为5.90,与NCBI网站中已公布的基因进行Blastp比对,发现该序列与香蕉炭疽菌(C.musae)、无花果炭疽菌(C.caricae)(登录号:GQ266389.1,GQ266386.1)的小柱孢酮脱水酶基因相似性分别为98%和97%。该基因的敲除载体pSCDGH-1已构建成功,为下一步获得SCD1基因敲除突变体,研究该基因功能打下了材料基础。

Colletotrichum gloeosporioides is one of the most serious diseases in Mango. In order to clarify the relationship between scytalone dehydratase gene SCD1 and pathogenicity of mango anthracnose （C. cloeosporioides）, SCD1 gene was cloned by homologous cloning, using the DNA of mango anthracnose as template. The sequence characteristics were analyzed, the conservative structure domains of protein were predicted, and the SCD1 gene knockout vector was constructed by the method of In-Fusion liD Cloning Kit. The results showed that the full DNA and cDNA length of SCD1 gene were 796 bp and 564 bp, together with two introns of 52 bp and 180 bp respectively, and encoded a putative protein of 187 amino acid with a molecular weight of 21.52 kD and isoelectric point of 5.90. The Blastp comparision between the gene released on NCBI and SCD1 gene indicated that the SCD1 sequence was highly similar （98% and 97%） to scytalone dehydratase SCD1 gene from banana anthracnose （C. musae） and fig anthracnose （C. caricae） （GQ266389.1, GQ266386.1）. The SCD1 gene knockout vector pSCD1GH-1 had been constructed successfully, which would lay a foundation for further the acquiring of knockout mutants and study on the genes function.