少孢节丛孢菌丝氨酸蛋白酶Aoz1基因启动子的克隆、鉴定及其功能研究

Sheep gastrointestinal nematodiasis is one of animal parasitic diseases, which is seriously harmful to the sheep industry. The wide use of chemical anthelmintics is still the main means to prevent and control the disease. However, with the increasing problems such as drug-resistant strains of insects, drug residues in animal foods and environmental pollution, it is urgent to seek a scientific way to deal with the relationship between control of animal diseases and environmental protection. The use of the natural enemies of nematode-trapping fungi and it's secreted extracellular protease is now considered a promising alternative for the biological control of gastrointestinal nematodiasis. Therefore, it necessary to explore molecular mechanisms on expression of nematode-trapping functional genes. On the basis of preliminary studies, representative of the nematode-trapping fungi-Arthrobotrys oligospora was studied in the project. 5' flanking regions of serine protease Aoz1 gene will be amplified by chromosomal DNA walking technique, then promoter and its regulatory sequences of the gene are analyzed and verified by series of deletion mutations and DNA gel retardation for clarifying it's molecular characteristics. The deleted and double promoter of the Aoz1 gene of recombinant Arthrobotrys oligospora were constructed using Agrobacterium tumefaciens mediated transformation (ATMT) technology, respectively, infection activity and genetic stability were analyzed in nematode. The functional of serine protease Aoz1 gene promoter of Arthrobotrys oligospora was further verified. The elucidation of the molecular mechanisms on expression regulation of Aoz1 gene will provide a scientific basis for development of biological agents with independent intellectual property rights to prevent and control sheep gastrointestinal nematodiasis.

绵羊消化道线虫病是严重危害养羊业生产的一类寄生虫疾病，目前使用化学驱虫药是防治该病的主要手段。然而，伴随着耐药虫株的不断产生、动物性食品的药物残留和环境污染等问题日益凸现，迫切需要寻求一种新的动物疫病-环境友好防控方法。利用线虫的自然天敌-捕食线虫性真菌及其分泌性胞外蛋白酶来防控动物线虫病是一种有应用前景的生物防控方法。本项目拟以捕食线虫性真菌的代表菌-少孢节丛孢菌为研究对象，利用染色体DNA步移技术扩增该菌丝氨酸蛋白酶Aoz1基因5'上游侧翼序列，通过系列缺失突变和DNA凝胶阻滞技术鉴定该基因启动子及顺式调控元件；利用根癌农杆菌介导的真菌转化技术（ATMT）构建Aoz1基因启动子缺失和双启动子的重组少孢节丛孢菌，分析重组菌Aoz1基因表达水平及捕食线虫活性，验证Aoz1基因启动子功能，揭示少孢节丛孢菌Aoz1基因表达调控的机制，为研发具有自主知识产权的绵羊线虫病生物防控制剂提供科学依据。

利用线虫的自然天敌-捕食线虫性真菌进行生物防治是防控动物线虫病的一种安全、有效的方法。丝氨酸蛋白酶是捕食线虫性真菌侵染线虫的一类重要酶类，在侵染线虫中发挥了重要作用。因此，利用分子生物学手段构建高效表达侵染性丝氨酸蛋白酶的菌株是研发捕食线虫活性生物制剂的关键。本课题以捕食线虫性真菌的代表菌-少孢节丛孢菌新疆分离株为研究对象，通过染色体DNA步移技术扩增少孢节丛孢菌丝氨酸蛋白酶Aoz1基因5'上游侧翼序列，用DNA凝胶阻滞技术对该基因启动子核心区域及其顺式调控元件进行鉴定，揭示了其分子特征；利用根癌农杆菌介导的真菌转化技术（ATMT）构建了Aoz1基因启动子缺失和双启动子的重组少孢节丛孢菌，并开展重组菌Aoz1基因表达水平及捕食线虫活性分析。结果成功扩增出别含有2806 bp、1980 bp、1489 bp、1120 bp、876 bp和662 bp 的Aoz1基因5'上游侧翼序列系列缺失片段，构建了6个含有EGFP 报告基因的少孢节丛孢菌Aoz1基因启动子报告载体。将构建的6个少孢节丛孢菌Aoz1基因启动子报告载体分别电转化少孢节丛孢菌分生孢子，证实含有Aoz1基因5'上游侧翼序列2806 bp、1980 bp、1489 bp、1120 bp和876 bp片段的重组载体转化的分生孢子在48-144h 后可发出黄绿色的荧光，表明克隆的Aoz1基因5'上游侧翼序列具有启动子活性。启动子预测分析证实，Aoz1基因5'上游侧翼2806 bp片段含有真菌顺式调控元件 CAAT-box、GC-box、TATA-box 和启动子通用元件CT-box，同时还含有Tubby、ZnCys_Ab0079、NF-X1_Pbl0001、Sp1、GCF、PEBP2、EIIF、element_II_rs-4、E2F、EivF/CREB 和Tubby等转录因子的结合位点。DNA凝胶阻滞分析表明，Aoz1基因5'上游侧翼序列含有转录因子的结合位点。同时，捕食活性分析结果表明，Aoz1基因双启动子重组少孢节丛孢菌株（BP）的捕食线虫活性显著高于少孢节丛孢菌母源菌株（NP）和Aoz1基因启动子缺失菌株（DP），提示成功构建了高效转录丝氨酸蛋白酶Aoz1的重组少孢节丛孢菌菌株。本研究完成了课题计划书中的所有任务，达到了研究预期目标，为研发具有自主知识产权的绵羊线虫病生物防控制剂提供了科学依据。