巴西橡胶树棒孢霉落叶病菌毒素基因cc004-cas克隆及致病作用

Cloning and Pathogenicity of Toxin Gene cc004-cas from Corynespora cassiicola on Hevea brasiliensis

为了探明多主棒孢菌(Corynespora cassiicola)毒素基因cc004-cas序列特征及其功能,以巴西橡胶树棒孢霉落叶病菌为材料,采用SEFA-PCR和RT-PCR技术对毒素基因cc004-cas进行扩增,并通过插入潮霉素B基因获得其突变体。序列特征分析结果表明,cc004-cas基因DNA和cDNA全长分别为2 800 bp和180bp,编码区含2个内含子(67 bp和49 bp),推测编码59个氨基酸,其分子量约为5.92 ku,等电点pⅠ为5.70,与NCBI中报道的C.cassiicola毒素基因cas编码的毒素前体pro-cassiicolin的氨基酸序列(0HABV25895.1)相似性达85%。cc004-cas突变体表型测定结果表明:在菌落和菌丝形态、生长,产孢量、孢子萌发,菌丝生长对温度、pH要求,产生漆酶和纤维素酶的能力方面与野生型无显著差异;但用菌饼接种橡胶树嫩叶,致病力略有下降,用粗毒素接种时,致病力明显减轻,可见毒素是多主棒孢菌的一个重要的毒力因子,但不是唯一的因子。

To reveal the sequence features and the function of a toxin encoding gene cc004-cas,cc004-cas gene was cloned from Corynespora cassiicola on Hevea brasiliensis by the method of SEFA-PCR and RT-PCR,and its knockout mutant was gained by inserting hygB gene into cc004-cas gene.Analysis of sequence characterization showed that the complete DNA and eDNA sequence of cc004-cas gene was 2 800 bp and 180 bp in size,respectively,with two introns of 67 bp and 49 bp,respectively,encoding a putative protein of 59-amino acid with a molecular weight of 5.92 ku and isoelectric point of 5.70.Analysis indicated that the putative protein of cc004-cas gene was homologous to C.cassiicola cas toxin gene coding product-pro-cassiicolin （0HABV25895.1）with a similarity of 85％ in NCBI.Phenotype changes of a cc004-cas gene mutant were tested and there were no obvious difference in the colony color,mycelia morphology and growth,sporulation quantity,conidial germination,requirement of temperature and pH,and laccase and ccllulase production between the mutant and the wild type.However the young leaves of the rubber tree inoculated with mycelia plugs produced typical symptoms but demonstrated weak virulence.While crude toxin was tested for pathogenicity,it displayed weaker virulence.That indicated cc004-cas toxin was a impotant virulence factor but not a unique factor in the pathogenicity of C.cassiicola in Hevea brasiliensis.