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为了明确内切葡聚糖酶CgEg1B与杧果炭疽病菌Colletotrichum gloeosporioides致病力的关系,本试验以杧果炭疽病菌DNA为模板,利用同源克隆技术扩增CgEg1B,对其序列特征、蛋白保守结构域进行分析,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1 077bp、1 020bp,编码区有内含子(57bp)1个,推测编码339个氨基酸,其分子量约为37.13kDa,等电点PI为5.11。与NCBI网站中已公布的基因进行Blastp比对,发现该序列与鳄梨炭疽菌C.gloeosporioides(EQB54542.1)的内切葡聚糖酶基因相似性为99%;该基因的敲除载体pCgEg1BGH-1构建成功。CgEg1B基因具备真菌内切葡聚糖酶基因家族的序列特征,推测CgEg1B基因可能与杧果炭疽病菌的侵入、定殖和致病性有关。
In order to clarify the relationship between the endoglucanase CgEg1B and pathogenicity of Colletotrichum gloeosporioides, we used the homologous cloning technique to amplify CgEg1B from the DNA of C. fortunei, and analyzed its sequence characteristics, conserved structure of protein Domain for analysis and construction of knockout vectors using the In-Fusion HD Cloning Kit technology. The results showed that the full-length cDNA and cDNA of this gene were 1 077bp and 1 020bp, respectively. The coding region contained 1 intron (57bp) and deduced 339 amino acids. The molecular weight of this gene was 37.13 kDa and the isoelectric point PI was 5.11. The Blastp alignment of the published genes in the NCBI website showed that the endoglucanase gene was 99% similar to C. gloeosporioides (EQB54542.1) in C. avium, and the knockout vector pCgEg1BGH -1 build is successful. The CgEg1B gene has the sequence characteristics of the fungal endoglucanase gene family. It is speculated that the CgEg1B gene may be involved in the invasion, colonization and pathogenicity of anthracnose.