Study On Mating Type Genes Of Asexual Spores From Cordyceps Militaris

Cordyceps militaris is a great market potential for development of medicinal fungi, however,in the process of artificial cultivation there are some problems, such as: strains degradation, non-fruiting or fruit body deformities, etc. These problems have hindered the development of Cordyceps militaris. At present the study on Cordyceps militaris is mainly focused on the choose of culture conditions, the active ingredient analysis and pharmacological research, the research about their basic biology, genetics and so on still weak,the genetic mechanism of strain degeneration research is empty.Four Cordyceps militaris homokaryon strains CM-28A,HJ-4,CM-23B,CM-H07 were used in this study and their single spore isolated strains were obtained. According to the sequences of Genbank database of mating type genes(Log No. AB084257 and AB194982), three specific primers were designed for amplifing mating type genes, MAT1-1-2、MAT1-1-1 and MAT1-2-1. Molecular identification on mating type genes of asexual spore isolated strains from Cordyceps militaris was carried by PCR, and the relationship between the lack of mating type and the normal formation of fruit bodies was analysised.At the same time ,study on morphology of asexual spore isolated strains was carried by optical microscope and scanning electron microscope.This paper’s main results were shown as following:1.Cordyceps militaris parental strains CM-23B and CM-H07 in this study contained two kinds of mating types genes , MAT-HMG(MAT1-2-1)and MAT-alpha(MAT1-1-1 and MAT1-1-2); Parental strains CM-28A and HJ-4 only contained MAT-alpha(MAT1-1-1and MAT1-1-2)mating type gene.2.Twenty single spore isolated strains were obtained from pateral strains CM-28A and HJ-4 respectively, they all contained the same mating type gene as their parental strains.3.Among100 signal spore isolated strains from parental strain CM-23B, 35 percents have the same mating type genes as their pateral strains; 40 percents only contained mating type gene, MAT- alpha(MAT1-1-1and MAT1-1-2),others contained mating type gene, MAT-HMG(MAT1-2-1). 4.100 signal spore isolated strains were obtained from parental strain CM-H07, among of these strains ,37 percents have the same mating type genes as their pateral strains; 33 percents only contained mating type gene, MAT- alpha(MAT1-1-1and MAT1-1-2),others contained mating type gene, MAT-HMG(MAT1-2-1).5.Asexual spores were found in the cultivation strains of mycelium, molecular identification on mating type genes of asexual spore isolated strains and their parental strains were carried by PCR. The results indicated that parental strain contained two kinds of mating type genes, while among the heterokaryon strains, a high proportion of signal spore isolated strains have single mating type gene, either MAT-alpha or MAT-HMG. It illustrated that mating type degradation was occurred in single spore isolates.6.The ability of Cordyceps militaris to form fruit body was carried ,the result indicated the typical pleomorphism of Cordyceps militaris, all no fruiting-body forming single spore isolation strains have single mating-type, either MAT-alpha or MAT-HMG., but all fruit body single spore isolation strains have both MAT-alpha and MAT-HMG.; Strains only contained mating-type MAT-HMG, have lost the color changing ability .7.The conidiogenous structure of natural strain CM-23B were obtained by optical microscope, it illustrated that the strain have two kinds of conidiogenous structure, paecilomyces-type and cephalosporin-type, but they do not grow on the same mycelium. Morphology of asexual spores was carried by scanning electron microscope. The result illustrated that most of asexual spores were spherical, a few oval, size 2-5μm..