香蕉突脐蠕孢叶斑病菌Brn1基因的PCR扩增与序列分析

摘要

[目的]为从分子水平进一步鉴定引起香蕉叶斑病的突脐蠕孢霉,对突脐蠕孢的Brn1基因序列进行了分析.[方法]用引物Brn1 01(5'-GCCAACATCGCAAACATGG-3’)and Brn1 02(5’-GCAAGCAGCACCGTCAATACCAAT-3')对3个分生孢子形态差异较大的香蕉突脐蠕孢叶斑病菌(CLER09 、D087和JL05)的Brn1基因进行PCR扩增和系统发育分析.[结果]不同菌株间存在简单的碱基缺失和替换,供试菌株与Exserohilum rostratum在系统发育树上聚类在同一个分支,与Exserohilum属其他种明显分开,形成一个独立的演化群.遗传距离分析表明,供试菌株与E.rostratum亲缘关系相近,菌株间的遗传距离为0.000～0.018,而Exserohilum属种间的遗传距离为0.020～0.093.[结论]E.rostratum菌株间的遗传变异较大,但仍属种内变异；Brn1基因可作为支持具有明显形态变异菌株为E.rostratum成员的分子依据.%[Objective]Different strains of Exserohilum rostratum,causing exserohilum banana leaf spot disease were characterized at molecular level using the conserved sequence of Brn1 gene.[Method] Brn1 gene of three pathogenic isolates (CLER09,D087 and JL05) of E.rostratum causing exserohilum banana leaf spot disease was amplified using primer pair Brn1 01 (5'-GCCAACATCGCAAACATGG-3') and Brn1 02 (5'-GCAAGCAGCACCGTCAATACCAAT-3'),and phylogenetic analysis was carried out.[Result] The results showed absence and substitute of single bases in Brn1 sequences among different isolates of E.rostratum isolated from diseased banana plant and sequence collected from GenBank database.The isolates collected from banana were clustered with E.rostratum in an absolute evolution group in the phylogenetic tree,and significantly distinguished from other species of Exserohilum spp.Analysis result of the genetic distance matrix showed that tested isolates were genetically close to E.rostratum sequences collected from GenBank database with intraspecies genetic distance of 0.000-0.018 and interspecies genetic distance of 0.020-0.093 in Exserohilum genus.[ Conclusion ] The obvious genetic difference among isolates of E.rostratum could be considered as intraspecific variation and the Brn1 gene sequence may be used for molecular evidence for supporting the isolates with markedly morphological difference as members of E.rostratum.