甘蔗梢腐病菌Fusarium sacchari CYP51基因克隆及遗传转化

Cloning and Genetic Transformation of Sugarcane Pokkah Boeng Fusarium sacchari CYP51 Gene

甾醇14α-去甲基化酶(CYP51)是生物细胞膜合成所需的一个非常重要的酶,在病原菌的耐药性、致病性和生长繁殖等方面发挥着非常重要的作用。研究表明干扰真菌的CYP51基因表达导致其无法正常生长,显著降低其致病性。本研究以甘蔗梢腐病菌甘蔗镰刀菌(Fusarium sacchari)为试验材料,根据基因组测序数据设计特异性引物克隆得到了FsCYP51基因全长和CDS全长。生物信息学分析表明,该基因序列全长1947 bp,编码区由2个内含子和3个外显子组成,CDS全长1554 bp,编码517个氨基酸,编码蛋白理论相对分子量为58.61 kDa。其编码蛋白的二级结构主要由α-螺旋和无规卷曲构成,具有典型的CYP51保守结构域。预测其亚细胞定位于细胞膜,且存在2个跨膜区域。系统进化分析表明,FsCYP51基因属于CYP51C这一类,与串珠镰刀菌(F.verticillioides)的CYP51C基因亲缘关系最近。同时,根据克隆到的基因全长和CDS全长构建了多价HIGS植物表达载体,并利用基因枪介导的遗传转化方法将干扰片段成功转化至甘蔗受体材料,为研究FsCYP51基因功能和创制抗梢腐病甘蔗种质奠定基础。

CYP51 is a very important enzyme for the biosynthesis of biological cell membrane,and plays a very important role in drug resistance,pathogenicity,growth and reproduction of pathogens.The interference with expression of CYP51 gene in fungi can prevent its normal growth and significantly reduce its pathogenicity.In this study,the full length of FsCYP51 gene and CDS were cloned by designing specific primers based on the genome sequencing data of Fusarium sacchari.Bioinformatics analysis showed that the total length of the gene was 1947 bp,and the coding region consisted of two introns and three exons.The total length of CDS was 1554 bp,encoding 517 amino acids.The theoretical relative molecular weight of the coding protein was 58.61 kDa.The secondary structure of the encoded protein was mainly composed ofα-helix and random coils,and it had a typical conserved domain CYP51.Its subcellular localization was predicted in the cell membrane and there were two transmembrane regions.Phylogenetic analysis showed that the FsCYP51 gene belonged to the CYP51C class,and was closely related to the CYP51C gene of F.verticillioides.At the same time,according to the full length of the gene and CDS,the polyvalent HIGS plant expression vector was constructed,and transformed into sugarcane by particle bombardment,which would lay a foundation for the study of function of FsCYP51 gene and the creation of transgenic sugarcane resistant to Pokkah boeng disease.