Gan Zhongwei, Yang Jinkui, Tao Nan, Liang Lianming, Mi Qili, Li Juan, Zhang Ke-Qin
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming 650091, People's Republic of China.
Appl Microbiol Biotechnol. 2007 Oct;76(6):1309-17. doi: 10.1007/s00253-007-1111-9. Epub 2007 Jul 31.
The nematophagous fungus Lecanicillium psalliotae (syn. Verticillium psalliotae) is a well-known biocontrol agent. In this study, a chitinase gene Lpchi1 was isolated for the first time from L. psalliotae using degenerate primers and DNA-walking technique. The cloned gene Lpchi1 encoding 423 amino acid residues shares a high degree of homology with other pathogenicity-related chitinases from entomopathogenic and mycoparasitic fungi. The complementary DNA sequence of the mature chitinase was amplified via reverse transcription polymerase chain reaction and expressed well in Pichia pastoris GS115. Through gel filtration, the recombinant chitinase was purified as a protein of ca. 45 kDa with an optimal activity at pH 7.0 and 37.6 degrees C. The purified chitinase LPCHI1 was found degrading chitinous components of eggs of the root-knot nematode Meloidogyne incognita and significantly influence its development. Moreover, our results also demonstrate that the protease Ver112 and the chitinase LPCHI1 from the same fungus interacted on the egg infection.
食线虫真菌双孢蘑菇轮枝菌（同义词：小菌核轮枝菌）是一种著名的生物防治剂。在本研究中，首次使用简并引物和DNA步移技术从双孢蘑菇轮枝菌中分离出几丁质酶基因Lpchi1。克隆的基因Lpchi1编码423个氨基酸残基，与来自昆虫病原真菌和真菌寄生真菌的其他致病性相关几丁质酶具有高度同源性。通过逆转录聚合酶链反应扩增成熟几丁质酶的互补DNA序列，并在毕赤酵母GS115中良好表达。通过凝胶过滤，重组几丁质酶被纯化为约45 kDa的蛋白质，在pH 7.0和37.6℃下具有最佳活性。发现纯化的几丁质酶LPCHI1可降解南方根结线虫卵的几丁质成分，并显著影响其发育。此外，我们的结果还表明，来自同一真菌的蛋白酶Ver112和几丁质酶LPCHI1在卵感染过程中相互作用。
