蛹虫草线粒体基因型快速检测体系的构建及线粒体遗传稳定性的初步研究

本研究的目的是建立一种快速确定蛹虫草菌株线粒体基因型的技术体系,并探讨蛹虫草连续传代培养后线粒体的遗传稳定性。从已知线粒体基因组的蛹虫草菌株中扩增线粒体内含子位点,将扩增产物混合并制作出两套DNA分子量标准,即在8个内含子位点分别具有内含子的8条扩增条带组成的M-I和在6个内含子位点分别缺失内含子的6条扩增条带组成的M-II。从待检测的蛹虫草菌株（包括3个已知和2个未知线粒体基因组的菌株）中扩增同样的（假定）内含子位点,然后通过琼脂糖凝胶电泳分别与制备好的两个DNA分子量标准进行比较,能够准确判断蛹虫草菌株的线粒体内含子分布模式,从而验证了所构建的线粒体基因型快速检测体系的有效性。选择10个蛹虫草组织分离菌株和8个单分生孢子菌株连续转接培养15代,没有发现线粒体内含子分布模式发生改变。本研究成功构建了快速检测蛹虫草线粒体基因型的技术体系,并发现蛹虫草线粒体具有很高的遗传稳定性,为开展蛹虫草线粒体遗传规律的研究奠定了基础。

Rapid detection of mitochondrial genotypes in Cordyceps militaris and preliminary investigation on mitochondrial genetic stability

The purpose of this study was to establish a technical scheme for rapid determination of mitochondrial genotypes of Cordyceps militaris strains, and to investigate the genetic stability of C. militaris mitochondria after successive subcultures. All known intron loci (eight in total) in C. militaris mitochondrial genomes were amplified from C. militaris strains with available mitochondrial genomes, and amplicons were mixed to establish two sets of DNA molecular weight standards (M-I and M-II). M-I consisted of eight fragments with each corresponding to one of the eight known introns; M-II consisted of six fragments with each missing one intron at six known intron loci. To test the effectiveness of the established detection system, the same (presumed) intron loci were amplified from C. militaris test strains, including three strains with available mitochondrial genomes and two strains without available mitochondrial genomes, and amplicons were compared with fragments in M-I and M-II by agarose gel electrophoreses. By this way, we could accurately determine the mitochondrial intron distribution pattern of a C. militaris strain, which verified the effectiveness of the established detection system. Finally, ten tissue strains and eight single spore strains of C. militaris were subcultured continuously for 15 generations, and distribution patterns of mitochondrial introns were not found to vary. This study successfully constructed the technique scheme for rapid detection of mitochondrial genotypes of C. militaris, and found that the mitochondria of C. militaris had high genetic stability, which laid a foundation for studying mitochondrial inheritance of the fungus.