Studies On The Structure,Evolution And Function Of MAT Genes In Orbiliaceae

Reproduction is one of the most basic characteristics of organisms,and it is the basis of biological adaptation and evolution.There are two kinds of reproduction including asexual reproduction and sexual reproduction.Fungi has become a research hotpot due to the extremely diverse reproductive styles,but so far little is known about the maintenance mechanism of sexual reproduction.The reproductive patterns,sexual reproductive mechanisms and pathways of these species,as well as the internal and environmental factors that affect sexual reproduction are still unclear.Orbiliaceae has become an ideal model for studying the mechanism of sexual reproduction because of its multiple reproductive patterns.Based on the separation and preservation of a variety of individual in Orbiliaceae that can carry out different reproductive modes,we analyze the structure,evolution and function of mating type genes to provide valuable clues for understanding the evolution and ecological adaptation of sexual reproduction of fungi.The main findings are as follows1.After all the genome sequencing,assembly and analysis,we have identified MAT idiomorphs of the genera Arthrobotrys,Drechslerella,Dactylella,Dicranididion.Orbilia,Dactylellina,15 strains included.The mating type genes were identified as 1445?4378 bp,encoding 434?1142 amino acids and 1?7 introns.At the same time,we indentified two homothallism species and two heterothallism species.2.The inter-and intraspecific sequence and structure analysis of mating type genes revealed that the genes have large polymorphisms and structure variance among species.The upstream genes of mating type genes are conserved,while the downstream genes have a large number of translocations and inversions.But the two proteins Alpha-box and HMG-box encode the same protein family,HMG-domain.Genetic analysis demonstrated that the selection pressures of mating genes is much greater than house-keeping genes.In different reproductive models,these genes are subjected to different selection pressures,MAT1?1 are identical but MATT-2 in the homothallism are far greater than heterothallism.We tentatively speculated MATT-1 was evolved from MAT1-2 based on these results.Phylogenetic studies on house-keeping genes and mating type genes indicate different phylogenetic tree topologies.This indicates that the mating type genes and the housekeeping genes have a large degree of differentiation,and it is highly probable that the mating genes are affected by greater selection pressure.In the class Dothideomycetes and Leotiomycetes.the difference in the phylogenetic topology is very obvious.3.Using Arthrobortrys oligospora as the research object,ITS,RPB2,?-tub.MAT1-1 and MAT1-2 were amplified from 96 strains from China.In the nature population,there are 47 strains of MAT1-1 and 49 strains of MATT-2,the ratio is close to 1:1.these dates indicate that this species is the possibility of sexual reproduction.We then divided all strains into two groups by Mating type locus.population genetics analysis,phylogenetic studies by single-gene and multi-gene were done with the two groups respectively.A.oligospore is mainly divided into three groups with obvious intraspecific differences.The phylogenetic trees that they were constructed by different genes present different topologies show that our samples are not clustering by geographic groups.STRUCTURE analysis shows that the MATT-1 is divided into four population;while the MAT]-2 is divided into two population,the entire A.oligospora we collected have population differentation,the intra-population contribution is greater than the inter-population in all groups.There is gene flow between populations,and multilocus linkage disequilibrium and phylogenetic incompatibility analysis indicate that there is random recombination within the population,and there may be sexual reproduction.Subsequently,we used a variety of media to induce sexual reproduction of the fungus under different conditions,and did not successfully induce its sexual reproductive structure.No recombination was found by sequencing.RNA was extracted and MAT1-1 gene was amplified for different induction periods.the transcription of the gene was found to suggest that sexual reproduction of the fungus may be controlled by a variety of factors.4.In order to further study the biological function of mating genes,we successfully constructed two knockout vectors of two mating genes in A.oligospora by electro-transformation and uracil-deficient Saccharomyces cerevisiae as carrier cells.Afterwards the ?MAT1-1 knockout strain was successfully obtained by CaC12-PEG mediated method.The growth rate,stress resistance,spore morphology and quantity of nematode captures of wild type and knockout strains were compared.The experimental results showed that there was no significant difference between the wild type and the knockout strains in terms of growth rate,stress resistance and ability to produce a trap,but statistical significance was found in the morphology and number of spores.