寄生于南方根结线虫卵的长梗木霉几丁质酶基因TlChi46的克隆

为进一步筛选高效寄生线虫真菌和阐明其寄生线虫卵的机理,本研究从湖北省烟草南方根结线虫雌虫分离到1株具高效生防潜力的菌株HBF1。形态学、rDNA-ITS和翻译延长因子tef1-α序列分析鉴定该菌为长梗木霉菌Trichoderma longibrachiatum,其对南方根结线虫卵第10 d寄生率为80.45%。通过简并引物设计和RACE技术克隆其几丁质酶基因,分析该基因序列及与其他几丁质酶的同源性。该菌是1株可以产生几丁质酶的南方根结线虫卵寄生真菌,第10 d几丁质酶的活性达到高峰,为24.88μmol/h/mL。本研究首次从长梗木霉HBF1菌株中克隆到1个几丁质酶基因TlChi46,该基因DNA全长1 793 bp,含3个内含子和1个1 272 bp的开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.23。同源性比对表明和昆虫寄生菌几丁质酶的亲缘关系较远。从长梗木霉HBF1中克隆得到的几丁质酶基因编码的几丁质酶的功能域可供进一步研究,高效产几丁质酶并有效寄生南方根结线虫卵的长梗木霉对南方根结线虫具有良好的生防潜力。

Cloning of a novel chitinase gene TlChi46 from Trichoderma longibrachiatum parasitizing on Meloidogyne incognita eggs

A fungus named HBF1 was isolated from female of M. incognita on tobacco in Hubei province, China and was identified as Trichoderma longibrachiatum based on morphological characteristics and molecular analysis of rDNA-ITS coupled with translation elongation factor 1-alpha. To identify virulent factors and elucidate the parasitic mechanism on M. incognita eggs, the egg parasitism of M. incognita eggs by T. longibrachiatum HBF1 was investigated in vitro. The percentage of parasitized eggs was 80.45% at the 10 th day after inoculation. From this isolate, the novel genomic and cDNA clones encoding chitinase have been cloned using the degenerate PCR primers and RACE techniques. The analysis of the gene and the deduced amino acid sequence along with its phylogeny analysis was by biology software. Results showed that HBF1 was able to penetrate nematode egg and produce chitinase, maximum activity of chitinase was 24.88 μmol/h/mL recorded at the tenth day after inocu-lation. We have cloned a novel chitinase gene TlChi46 from T. longibrachiatum HBF1 for the first time. The gene is 1 793 bp in length and contains three putative introns. The ORF of TlChi46 is 1 272 bp in size with encoding protein of 423 aa , molecular mass of 45.9 kDa and pI of 5.23. Phylogeny analysis reveals that this chitinase and other reported Trichoderma spp. chitinases are clustered together and are phylogenetically distant from other entomopathogenic fungi. These results demonstrated that the amino acid sequence coded by chitinase gene TlChi46 from T. longibrachiatum HBF1 possesses promising functional domains for the further research, T. longibrachiatum HBF1 with the the strong chitinolytic and egg-parasitic activity has a great biocontrol potential against M. incognita.