橡胶树胶孢炭疽病菌CgBASP2基因敲除突变体构建及其致病力分析

Pathogenicity analysis and construction of CgBASP2 knockout mutant of Colletotrichum gloeosporioides infecting Hevea brasiliensis

在前期对橡胶树胶胞炭疽病菌分泌蛋白组进行预测的基础上,通过RT-PCR的方法克隆了1个候选分泌蛋白基因并命名为Cg BASP2(Biotrophy-associated Secreted Proteins 2 of Colletotrichum gloeosporioides)。该基因编码100个氨基酸,相对分子质量约为9.85×103。生物信息学分析结果表明,该蛋白含有1个18氨基酸的信号肽序列,不含任何跨膜结构域。氨基酸序列比对结果表明,Cg BASP2与草莓胶孢炭疽菌(Colletotrichum gloeosporioides Nara gc5)、西瓜炭疽菌(Colletotrichum orbiculare MAFF)、玉米炭疽菌(Colletotrichum graminicola)、菜豆炭疽菌(Colletotrichum higginsiaum)和稻瘟菌(Magnaporthe oryzae 70-15)的BASP2同源,同源性分别为100%,90%,87%,87%和67%。为了研究该基因的功能,本研究通过同源重组技术构建了橡胶树胶孢炭疽病菌Cg BASP2基因的敲除突变株ΔCg BASP2。进一步的分析发现,与野生型菌株相比ΔCg BASP2突变菌株不能对橡胶树叶片产生致病性,由此可见,Cg BASP2基因在胶胞炭疽病菌的致病过程中起重要作用。

Based on the prediction result of secreted proteome of Colletotrichum gloeosporioides infecting rubber tree（ Hevea brasiliensis）,one of the candidate genes was cloned by RT-PCR and named as Biotrophy- associated Secreted Protein 2 of C. gloeosporioides（ Cg BASP2）. This gene was sequenced and found to encode 100 amino acids with a relative molecular weight of about 9. 85 × 103. Bioinformatics analysis showed that the gene contains an 18 aa signal peptide at N terminal,but without any transmembrane domain. Alignment analysis showed that the amino acid sequence of the gene Cg BASP2 from H. brasiliensis is 100%,90%,87%,87% and 67%similar to that of the Cg BASP2 from Colletotrichum gloeosporioides Nara gc5,Colletotrichum orbiculare MAFF,Colletotrichum graminicola,Colletotrichum higginsiaum and Magnaporthe oryzae 70- 15,respectively. The CgBASP2 gene knockout mutant strain ΔCg BASP2 was constructed by using homologous recombination to study its biological functions. This ΔCg BASP2 mutant strain inoculated onto the leaves of rubber tree was found to lose pathogenicity while the wild type strain had good pathogenicity. This result implied that the Cg BASP2 gene might probably play an important role in the pathogenicity of C. gloeosporioides.