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
发布时间：2019-01-19 10:44
【摘要】：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~汶 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。
[Abstract]:Mango anthracnose (Colletotrichum gloeosporioides) is one of the important diseases of mango. In order to clarify the relationship between SCD1 gene and pathogenicity of mango anthracnose, DNA of mango anthracnose was used as a template in this study. Homologous cloning technique was used to amplify SCD1, to analyze its sequence characteristics. The conserved domain of its protein was speculated, and the knockout vector was constructed by means of In-Fusion~ HD Cloning Kit technique. The results showed that the full length of DNA and cDNA were 796 bp,564 bp, respectively. There were two introns (52 bp,180 bp), in size) encoding 187 amino acids, the molecular weight of which was about 21.52 kD, isoelectric point PI was 5.90. Blastp alignment with genes published on the NCBI website revealed that the sequence was linked to banana anthrax (C.musae) and fig anthrax (C.caricae) (accession number: GQ266389.1,) The similarity of cyclosporine dehydratase gene in GQ266386.1 was 98% and 97%, respectively. The knockout vector pSCDGH-1 has been successfully constructed, which lays the material foundation for the next step to obtain the mutant of SCD1 gene knockout and to study the function of the gene.
【作者单位】： 海南大学环境与植物保护学院海南大学热带作物种质资源保护与开发利用教育部重点实验室;中国热带农业科学院环境与植物保护研究所农业部热带作物有害生物综合治理重点实验室;
【基金】：国家自然科学基金(31460455) 公益性行业(农业)科研专项项目 芒果产业技术研究与示范(201203092-2)共同资助
【分类号】：S436.67
，
本文编号：2411295
[Abstract]:Mango anthracnose (Colletotrichum gloeosporioides) is one of the important diseases of mango. In order to clarify the relationship between SCD1 gene and pathogenicity of mango anthracnose, DNA of mango anthracnose was used as a template in this study. Homologous cloning technique was used to amplify SCD1, to analyze its sequence characteristics. The conserved domain of its protein was speculated, and the knockout vector was constructed by means of In-Fusion~ HD Cloning Kit technique. The results showed that the full length of DNA and cDNA were 796 bp,564 bp, respectively. There were two introns (52 bp,180 bp), in size) encoding 187 amino acids, the molecular weight of which was about 21.52 kD, isoelectric point PI was 5.90. Blastp alignment with genes published on the NCBI website revealed that the sequence was linked to banana anthrax (C.musae) and fig anthrax (C.caricae) (accession number: GQ266389.1,) The similarity of cyclosporine dehydratase gene in GQ266386.1 was 98% and 97%, respectively. The knockout vector pSCDGH-1 has been successfully constructed, which lays the material foundation for the next step to obtain the mutant of SCD1 gene knockout and to study the function of the gene.
【作者单位】： 海南大学环境与植物保护学院海南大学热带作物种质资源保护与开发利用教育部重点实验室;中国热带农业科学院环境与植物保护研究所农业部热带作物有害生物综合治理重点实验室;
【基金】：国家自然科学基金(31460455) 公益性行业(农业)科研专项项目 芒果产业技术研究与示范(201203092-2)共同资助
【分类号】：S436.67
，
本文编号：2411295
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