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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 important diseases of mango. In order to clarify the relationship between the cerealosporidium dehydratase gene SCD1 and the pathogenicity of the pathogenic fungi, the present study used the DNA of mango as a template, SCD1 was amplified by homology cloning technique, its sequence characteristics were analyzed, the protein conserved domain was deduced, and knock-out vector was constructed by In-Fusion ~ HD Cloning Kit technology. The results showed that the full-length cDNA and cDNA of this gene were 796 bp and 564 bp respectively. There were two introns in the coding region (size 52 bp and 180 bp). The deduced cDNA encoded 187 amino acids with a molecular weight of about 21.52 kD. The PI of the locus was 5.90, which was compared with the published genes of NCBI by Blastp. The sequence was found to be closely related to C. musae and C.caricae (accession number: GQ266389.1, GQ266386). 1) had a similarity of 98% and 97%, respectively. The gene knockout vector pSCDGH-1 has been successfully constructed, which provides a material basis for further study on the function of this gene by obtaining the SCD1 knockout mutant in the next step.