运用基因枪法进行蛹虫草遗传转化的研究

采用蛹虫草Cordyceps militaris（L.）Link]野生型菌株CM01为供试材料，以金粉包裹质粒pDHt-gpdA-GFP-bar，运用基因枪法转化，经草铵膦抗性筛选，最终在靶距离6 cm或9 cm，氦气压力7.58 × 106 Pa或8.96 × 106 Pa条件下，获得2个遗传稳定的转化子Gfp2与Gfp3，转化效率为0.4 cfu ? μg-1。PCR鉴定与Southern杂交分析显示，草铵膦抗性基因Bar已经以单拷贝或者多拷贝方式整合到蛹虫草转化子的基因组中。蛹虫草转化子的菌丝在荧光显微镜下可以观测到绿色荧光，表明载体携带的绿色荧光蛋白基因在蛹虫草转化子中得到表达。研究结果表明可以把基因枪法应用于真菌蛹虫草，建立了一种简便有效的遗传转化方法，而转化效率需要通过优化基因枪参数设置、受体材料制备等途径进一步提高。

Effective Transformation of Cordyceps militaris by Particle Bombardment

An effective and convenient transformation method of Cordyceps militaris were established by means of particle bombardment. Coating microcarriers with plasmid pDHt-gpdA-GFP-bar，we transferred C. militaris CM01 by particle bombardment. After being selected by glufosinate-ammonium，two transformants were obtained under three conditions differing in the two parameters of target distance （6 cm or 9 cm） and helium pressure（7.58 × 106 Pa or 8.96 × 106 Pa）. The transformation frequency was 0.4 cfu? μg-1. PCR and southern blot results indicated that the selective marker gene Bar was integrated into the genomic DNA，a single copy or multiple copies integration，and genetically stable. Green fluorescence of the C. militaris transformant mycelia could be observed under fluorescent microscope，which indicated that the expression of green fluorescent protein was successful in the C. militaris transformants. Transformation by particle bombardment enriched the transformation methods of C. militaris. However，sample preparation and parameter setting need to be optimized，in order to improve the efficiency of transformation.