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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。
In order to further screen the efficient parasitic nematode fungi and elucidate the mechanism of its parasitic nematode eggs, a strain HBF1 with high potential of biocontrol potential was isolated from the tobacco root-knot nematode in Hubei Province. Morphology, rDNA-ITS and tef1-α sequence analysis showed that Trichoderma longibrachiatum was identified as Trichoderma longibrachiatum, and the parasitism rate was 80.45% on the 10th day of Southern root knot nematode eggs. The chitinase gene was cloned by degenerate primer design and RACE technology. The sequence of the chitinase gene and its homology with other chitinase genes were analyzed. The fungus is a parasitic fungi of M. incognita which can produce chitinase. The chitinase activity peaked at 10 d, which was 24.88 μmol / h / mL. In this study, for the first time, a chitinase gene TlChi46 was cloned from Trichoderma longibrachiatum HBF1. The full-length DNA of this gene is 1 793 bp in length, contains 3 introns and 1 open reading frame of 1 272 bp encoding 423 Amino acids, theoretical molecular weight 45.9 kDa, isoelectric point 5.23. Homology comparison indicated that the genetic relationship with the insect parasite chitinase is far. The functional domain of the chitinase encoded by the chitinase gene cloned from Trichoderma longibrachiatum HBF1 is available for further investigation. The Trichoderma longibrachiatum that efficiently produces chitinase and effectively parasitizes southern root-knot nematode Southern root knot nematode has good potential of biological control.