樟树溃疡病菌糖苷水解酶基因LtGH88的克隆与表达

Cloning and Expression of LtGH88 in Lasiodiplodia theobromae

【目的】可可毛色二孢(Lasiodiplodia theobromae)是樟树溃疡病的主要致病菌。糖苷水解酶是病原菌与寄主植物互作过程中的关键因子。对可可毛色二孢糖苷水解酶基因LtGH88进行克隆与表达分析,为研究LtGH88在樟树溃疡病菌致病机制中的作用奠定基础。【方法】采用PCR方法从可可毛色二孢侵染樟树枝干组织中获得LtGH88基因的CDS序列,运用生物信息学方法分析预测编码蛋白的特征和功能,通过qRT-PCR测定LtGH88在可可毛色二孢侵染樟树过程中的表达量;再利用农杆菌介导的烟草瞬时表达技术初步分析LtGH88基因的功能。【结果】LtGH88基因编码序列长度为1152 bp。其编码蛋白相对分子质量约为42.8 kDa,等电点(pI)为4.56,二级结构由49.09%的α螺旋、11.23%的延伸链和34.99%的无规则卷曲组成。蛋白N端包含18个氨基酸组成的信号肽,属于糖苷水解酶88(GH88)家族,能酶解果胶物质。LtGH88基因在樟树溃疡病菌侵染早期显著表达,不引起本氏烟叶片细胞坏死,且可抑制Bax引发的烟草叶片过敏性坏死反应。【结论】糖苷水解酶LtGH88可能通过抑制寄主免疫反应促进溃疡病菌可可毛色二孢在樟树枝干中的侵入和定殖。

【Objective】The glycoside hydrolase gene LtGH88 of Lasiodiplodia theobromae was cloned to study the mechanism of canker in Cinnamomum camphora,which is the major pathogen that causes the disease.【Methods】The LtGH88 encoding sequence was cloned by PCR from tissues of C.camphora infected by L.theobromae to determine the characteristics and functions of the protein by bioinformatic methods.Expression of the gene was detected by real-time quantitative PCR,and functions analyzed using the Agrobacterium tumefaciens mediated transient transformation in Nicotianaben thamiana.【Results】The full open reading frame of LtGH88 was 1152 bp with a molecular weight of 42.8 kDa and a theoretical isoelectric point of 4.56.The predicted secondary structure of the protein consisted of 49.09%αhelix,11.23%extended chain,and 34.99%random coil.A signal peptide of 1-18 amino acids was located at the N terminus.The protein belonged to the glycoside hydrolase family 88(GH88)capable of degrading pectin.LtGH88 was significantly expressed in the early stage of the infection.It did not cause cell necrosis in the leaf of N.thamiana but was able to inhibit the hypersensitive response(HR)induced by Bax.【Conclusion】It was postulated that LtGH88 in L.theobromae inhibited the immune response of C.camphora facilitating the pathogenic invasion and colonization on the host plant.