农杆菌介导的蛹虫草营养缺陷型菌株和遗传转化体系的构建

Construction of Agrobacterium-mediated auxotrophic strain and genetic transformation system of Cordyceps militaris

摘要:

【背景】天然蛹虫草是蛹虫草菌侵染昆虫蛹或虫形成的子实体，具有非常重要的生物药理活性。目前蛹虫草基因组测序已经完成，但是其分子生物学研究较少。【目的】在蛹虫草中构建一种以尿苷/尿嘧啶营养缺陷型为筛选标记的农杆菌介导的转基因体系。【方法】乳清酸核苷-5'-磷酸(orotidine-5'-monophosphate，OMP)脱羧酶为尿嘧啶合成必需酶，利用根癌农杆菌介导的转化(Agrobacterium tumefaciens-mediated transformation，ATMT)方法，通过同源重组对蛹虫草野生型菌株中该酶的编码基因pyrG进行敲除，构建尿苷/尿嘧啶营养缺陷型突变体。然后，利用本实验室已有的米曲霉pyrG为筛选标记的二元转化载体，通过农杆菌转化法对该营养缺陷型菌株进行遗传转化。【结果】通过同源重组法，成功敲除pyrG构建了尿嘧啶营养缺陷型蛹虫草，以此为背景，在22 ℃共培养66 h，成功对蛹虫草实现转基因，转化效率为(75±35)/10⁶孢子。另外，本研究还发现丝状真菌常用的构巢曲霉3-磷酸甘油醛脱氢酶启动子PgpdA及α淀粉酶启动子PamyB不能在蛹虫草中驱动GFP或DsRed报告基因表达，而蛹虫草自身延伸因子的启动子IF则能很好地表达GFP荧光报告基因。【结论】本研究构建了一种以尿嘧啶营养缺陷型为筛选标记的农杆菌介导的转基因体系，为研究蛹虫草重组表达和基因功能提供了一个有前景的基因操作工具。

Abstract:

[Background] Natural Cordyceps militaris is the fruiting body formed by Cordyceps militaris infects insect pupae or insects and has crucial biological and pharmacological activities. At the moment, the genome of C. militaris has been sequenced, but the research in molecular biology is limited. [Objective] To construct the Agrobacterium tumefaciens-mediated transformation (ATMT) system in C. militaris with uridine/uracil auxotrophic gene as the selection marker. [Methods] Orotidine-5'-monophosphate (OMP) decarboxylase is an essential enzyme for uracil synthesis. By using ATMT, We first knocked out the gene encoding pyrG of OMP decarboxylase in wild C. militaris by homologous recombination, and then constructed the uridine/uracil auxotrophic mutant. Afterward, the pyrG-carrying binary vector of Aspergillus oryzae was used to construct the complementary strain by ATMT. [Results] The pyrG was successfully knocked by homologous recombination and the uridine/uracil auxotrophic mutant was constructed. Under this background, the transformation efficiency reached (75±35)/10⁶ conidia after co-culture for 66 h at 22 ℃. In addition, the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter PgpdA and α-amylase promoter PamyB, which are commonly used by filamentous fungi, failed to drive the expression of GFP or DsRed reporter gene in C. militaris, but the promoter IF of C. militaris extension factor could well express GFP. [Conclusion] We constructed an ATMT system with uracil auxotrophic as a selection marker, providing a promising genetic tool for research on recombinant expression and gene function of C. militaris.