Mating Type Genes Cloning And Its Impact On The Formation Of Fruit Bodies Of The Cordyceps Militaris

Cordyceps militaris has useful medicinal applications.lt is a potential source of bioactive molecules and 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 C. militaris artificial cultivation.At present the study on C. 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 is still weak,the research on genetic mechanism of strain degeneration is blank.In this study, the homokaryon strains a4,a3,a7,a8as well as other C. militaris strains were used, specific primers were designed according to the sequences of mating type genes for PCR, combined with genomic walking,we obtained3C. militaris mating type genes MAT1-1-1, MAT1-1-2and MAT1-2-1. MAT1-1-1and MAT1-1-2’s mating type is alpha, MAT1-2-1’s mating type is HMGAt the same time,we preliminary analysised the relationship between the lack of mating type and the formation of fruit bodies and studied on the mating type of the strains which were germinated by asexual spores of C. militaris.The main results were as follows:1. From homokaryon strains a4and a7,we obtained3C. militaris mating type genes MAT1-2-1, MAT1-1-1and MAT1-1-2. The DNA sequences of these mating type genes with the similar mating type genes (accession number AB084257and AB194982) on NCBI have high similarity (>89%). MAT1-1-1full-length sequences (780bp) were first reported.2. We detected the mating type genes of the fruiting and non-fruiting strains of the same species,using specific primers which designed according to the DNA sequences of the C. militaris mating type genes. The result indicated that the fruiting strains contained two kinds of mating type MAT-HMG (MAT1-2-1) and MAT-alpha (MAT1-1-1and MAT1-1-2), but non-fruiting strains contained only one of them. This showed that the non-fruiting strains lost one of the mating type. This result provided experimental basis for use of the molecular biology methods to test the strains whether they could be applied to production.3. Asexual spores were found in the mycelium of the C. militaris cultivation strains,we isolated the asexual spores and obtained10isolated strains.The mating type genes of these isolated strains and parental strain were detected by specific PCR. The result indicated that parental strain contained two kinds of mating types, while6single spore isolates contained only one of them. This showed that mating type deletion was occurred in single spore isolates, which can be inferred the production of asexual spores could be one reason for the degradation of C. militaris strain.