基于CRISPR/Cas9的长枝木霉T6菌株PRA1基因杀线分子调控机制解析

Heterodera avenae is a class of widely and commonly occurred plant pathogenic nematodes in cereal crop growing areas that severely restricted the production of wheat in China. Thus, biological control was considered as one of the hot spots in the world. Trichoderma longibrachiatum T6 is a well-known multifunctional bio-control fungus against several plant pathogens with an independent intellectual property right. In our previous studies, we discovered the strain of T. longibrachiatum T6 exhibited significantly nematicidal activity against the plant pathogenic nematodes by the morphological, physiological and biochemical, and metabolomics level. However, the molecular mechanism of the nematicial activity of T. longibrachiatum T6 against Heterodera avenae is still unclear. In the present project, the strain of T. longibrachiatum T6 and the cereal cysts nematodes of H. avenae will considered as the experiment materials to (i) clone the full-sequence cDNA of acidic serine protease gene (PRA1) from T. longibrachiatum T6, and analyze its expression characteristics by the methods of RACE and qRT-PCR, respectively; (ii) establish the CRISPR/Cas9 editing system for T. longibrachiatum T6 strain; (iii) construct the gRNA expression vector for PRA1 gene and obtain the strains of knockout mutant, and (iv) analyze the function of nematicidal gene by CRISPR/Cas9 system and explore its nematicidal mechanism by the molecular level. The purpose of our present project will clarify the nematicidal mechanism of T. longibrachiatum T6 against Heterodera avenae by the molecular level. Our results will provide the significance in improving the Trichoderma spp. function, and also provide the theoretical and practical significance in developing the new types and high efficient microbial nematicide agents in the future.

小麦禾谷孢囊线虫的普遍发生严重制约了我国小麦的生产，开展其生物防治已成为国内外研究的热点之一。长枝木霉T6是一株具有自主知识产权的多功能生防菌株。前期从形态学、生理生化和代谢组学等水平初步证实了长枝木霉T6菌株对植物病原线虫具有较强的毒杀作用，但有关其杀线分子机制尚未清楚。本研究将以长枝木霉T6菌株和禾谷孢囊线虫为试材，(i)利用RACE和qRT-PCR技术克隆T6菌株酸性丝氨酸蛋白酶基因 (PRA1) 的cDNA全长序列并明确其表达特性；(ii)建立T6菌株CRISPR/Cas9编辑系统；(iii)构建PRA1基因gRNA表达载体，获得PRA1基因定点敲除突变体；(iv)利用构建的CRISPR/Cas9系统分析T6菌株杀线功能及解析其分子机制等研究，旨在从分子水平上揭示T6菌株对禾谷孢囊线虫的毒杀作用机制，研究结果对于杀线木霉菌株的改良和新型微生物源农药的研发具有重要的理论和实践价值。

小麦禾谷孢囊线虫的普遍发生严重制约了我国小麦的生产，开展其生物防治已成为国内外研究热点之一。本研究通过对长枝木霉T6菌株PRA1基因克隆及表达特性分析、CRISPR/Cas9敲除系统建立、PRA1基因定点敲除突变体鉴定及其杀线功能和作用机制解析等研究。研究结果表明，克隆获得了编码T6菌株酸性丝氨酸蛋白酶活性的PRA1-1和PRA1-2基因cDNA全长序列，其长度分别为1171 bp和1100 bp；探明了T6菌株T6-PRA1-1和T6-PRA1-2基因生物信息学特点，并明确了接种孢囊后T6-PRA1-1和T6-PRA1-2基因表达量呈上调表达趋势，且随着接种时间增加，其相对表达量呈现出增加-降低-增加趋势；克隆了T6-Cas9基因及优化了其密码子，建立了T6菌株T6-CRISPR/Cas9编辑系统，并构建了PRA1基因sg RNA表达盒及体外转录和转化了sg RNA 表达载体，共获得7株T6菌株突变体；鉴定了T6 菌株PRA1基因功能，发现其能够编码和调控菌株胞外丝氨酸蛋白酶活性和含量，进而提高T6菌株对禾谷孢囊线虫孢囊的寄生和毒杀作用效果，显著降低孢囊的孵化率；优化了PT6蛋白培养滤液最佳杀线条件为pH 6.64、温度26.52℃和时间6.8天，在此条件下其杀线活性高达86.10%，并明确了PT6蛋白培养滤液对禾谷孢囊线虫2龄幼虫具有较强的毒杀作用，且稳定性较好；建立了T6菌株胞外杀线蛋白分离纯化方法，并基于质谱分析鉴定了PT6蛋白种类和功能，以及构建了T6菌株T6-GFP标记菌株，并明确了其对线虫寄生和致死作用效果及PRA1基因瞬时表达情况；揭示了T6菌株对线虫寄生和毒杀分子作用机制主要通过PRA1基因编码和调控菌株胞外丝氨酸蛋白酶活性和分泌，提高菌株对孢囊或2龄幼虫的寄生和毒杀作用活性，降低孢囊的孵化率或2龄幼虫的活性。研究成果对于揭示T6菌株对禾谷孢囊线虫的寄生和毒杀分子作用机制、木霉杀线菌株的改良和新型微生物源农药的研发具有重要理论和实践价值。项目实施期间先后发表高质量论文16篇 （其中SCI论文7篇，单篇最高影响因子8.025，累计影响因子32.822，中文核心期刊9篇），待发表论文3篇；申请国家专利10件，其中授权国家专利6件，受理4件；培养青年教师1名，研究生6名。