橡胶树胶孢炭疽菌效应蛋白基因CgE35对孢子产量的影响

The Effect of Effector Protein Gene CgE35 on Conidia Production of Colletotrichum gloeosporioides in Hevea brasiliensis

橡胶树炭疽病是橡胶树的重要真菌病害之一,主要由胶孢炭疽菌引起。效应蛋白是病原菌侵染植物的重要致病因子,在寄主与病原菌的相互识别中发挥重要作用。为了进一步阐明橡胶树炭疽菌的致病机理,课题组前期对橡胶树胶孢炭疽菌的效应蛋白在基因组水平上进行了预测,其中一个编码候选效应蛋白的基因被命名为CgE35。本研究对CgE35基因进行了扩增,并对其所编码的效应蛋白CgE35进行了生物信息学的分析,分析结果表明,该基因编码一条由200个氨基酸组成的多肽,分子质量约为21.7 kD,其氨基段含有一段由24个氨基酸组成的典型信号肽序列,且该蛋白不含任何跨膜结构域和已知的保守结构域。为了进一步研究该基因的功能,我们根据同源重组原理,构建了 CgE35基因的敲除载体pCB1532-CgE35,通过PEG介导的原生质体转化法将其转入胶孢炭疽菌原生质体细胞中进行同源交换,经过对转化子进行抗性筛选和分子鉴定,获得CgE35基因的敲除突变体ACgE35,并对其孢子产量和菌落生长情况进行分析,发现胶孢炭疽菌CgE35基因的敲除突变体ACgE35的孢子产量明显低于野生型菌株,而二者的菌落生长情况没有差异。以上结果表明,橡胶树胶孢炭疽菌CgE35基因编码一个未知功能的分泌蛋白,该基因与胶孢炭疽菌的产孢能力有关,但与其菌丝的生长关系不大。本研究结果为阐明胶孢炭疽菌的致病机理,建立新型橡胶树炭疽病防控策略提供理论依据。

Anthracnose is one of the most important fungal diseases of rubber tree mainly caused by Colletotrichum gloeosporioides. Effectors are key pathogenic factors of pathogen and function in fungal-plant interactions. In order to further elucidate the pathogenic mechanism of Colletotrichum gloeosporioides, in this study, a candidate gene encoding candidate effector was predicted and named as CgE35. In this study, CgE35 gene was cloned by RT-PCR and analyzed by bioinformatics approaches. Bioinformatics analysis showed that CgE35 encoded a 200 amino acid polypeptide with a molecular mass of 21.7 kD. CgE35 protein has a 24 aa signal peptide at N-terminal, but does not contain any trans-membrane domain and any known conserved domain. In order to study further on the function of this gene and based on the principle of homologous recombination the knockout vector of CgE35 gene, pCB1532- CgE35, was constructed and transformed into the hypha protoplast of Colletotrichum gloeosporioides by PEG-mediated transformation. After homologous exchange, the chlorimuron ethyl-resistant transformants were confirmed by PCR, the Colletotrichum gloeosporioides knock-out mutant of CgE35 gene, 4CgE35, was generated. Then the fungal growth rate and conidiation ability of ACgE35 were analyzed. The conidia production of ACgE35 was reduced to less than a-half that of the WT and there were no significant differences in the growth speed of aerial hyphae between the WT and ACgE35 strains. The results above show that, CgE35 protein is an unknown secreted protein. The CgE35 gene contributes to the ability to produce conidia but not colony growth of Colletotrichum gloeosporioides. The results of this study provide a theoretical basis for elucidating the pathogenic mechanism of Colletotrichum gloeosporioides and establishing a new rubber tree anthracnose prevention and control strategy.