Preliminary Study On Expression Features Of The Mating-type Genes And The Interaction Factors Of MAT1-1-3 In Villosiclava Virens

Sexual reproduction process is closely related to infection cycle in Villosiclava virens,and sclerotium plays an important role in the sexual reproduction process.While its sclerotium formed in the surface of false smut ball,clarifying the function and importance of false smut balls in sexual reproduction process is necessary to provide evidence for revealing the infection cycle of Villosiclava virens.The sexual reproduction process of Villosiclava virens is regulated by mating type idiomorphs.To preliminarily analyze the function and importance of mating type genes,the expression of mating type genes in four key processes of sclerotium germination were detected.In addition,screening MAT1-1-3 strong interaction factors by yeast two-hybrid may contribute to reveal the regulatory mechanism and signaling pathway of sexual reproduction in V.virens.1.It was proved that Villosiclava virens is a heterothallic fungus and its sclerotium formed in the surface of false smut ball plays an important role in the process of sexual reproduction.Mating type idiomorphs are the genetic basis of cross compatibility and sexual reproduction in V.viren.In order to clarify the function and importance of false smut balls in sexual reproduction of V.virens,649 false smut balls without sclerotia collected from 9 areas in Jiangsu and Hunan Province were detected to identify the mating type and the fertility.Of the 218 rice false smut balls which were employed to detected mating type,95 rice false smut balls only contained MAT1-1 mating-type hyphae,accounting for 43.58%;94 rice false smut balls only contained MAT1-2 mating-type hyphae,accounting for 43.12%;29 rice false smut balls contained both MAT1-1 and MAT1-2 mating-type hyphae,accounting for 13.3%.24 of the 431 rice false smut balls were employed in the fertility assay could germinate,while they were not found forming the sexual progenies ultimately.The subsequent inoculation assay further proved that false smut balls still played an important role in the genetic variation of V.virens as a part of the false smut balls had the material basis for the formation of ascospores,although the germination rate of the rice false smut balls without sclerotia were very low and they had not been observed to finish the sexual reproduction.2.Sclerotia formed in the rice that two different mating-type Ustilaginoidea virens were inoculated to were collected to germinate.Four key different stages were defined in 70th day of the germination process.Specific primers were designed based on the cDNA sequence of mating type genes(MAT1-1-1,MAT1-1-2,MAT1-1-3 and MAT1-2-8).Using a-tubulin-2 as a reference gene and the corresponding vegetative propagule as the control check,we performed the qRT-PCR to analyzed the expression of mating type genes in the four processes of sclerotium germination with the method of 2-△△CT.The results showed that the expression of mating type gene in four key stages of sclerotia germinate were much lower than that of propagule in vegetative stage,indicating that these genes may serve as negative regulators in the sexual reproduction.In addition,the expression of MAT1-1-3 and MAT1-2-8 had significant differences in the four different stages,thus they should be very important during the sexual reproduction in V.virens.3.The total RNA was extracted from the U.virens P1,then dsDNA were amplified by SMART technology and LD-PCR whose length ranged from 250 bp to 5000 bp.By transforming cDNA and pGADT7-Rec into competent yeast strain Y187,qualified cDNA library of U.virens was constructed successfully.According to the Matchmaker Gold Yeast Two-Hybrid System,the pGBKT7-MAT1-1-3 without toxicity and self activation was regarded as the bait to screen strong interaction factors of MAT1-1-3 in the cDNA library of Pl.At last total 61 positive clones were purified and acquired in the defective medium SD/-Ade/-His/-Leu/-Trp/X-a-GaI/AbA,of which 46 were sequenced successfully and analyzed.In the end,7 candidate proteins interacting with MAT1-1-3 were identified,including the Putative O-methyltransferase,Putative SUMO ligase SizA,Type Ⅱ restriction M6 adenine DNA methyltransferase or TATA box-binding,protein-associated factor,Eukaryotic translation initiation factor 3 subunit 6,60S acidic ribosomal protein P2,Pre-mRNA splicing factor and Ankyrin repeat protein.The determination of interaction factors of MAT1-1-3 provided the basis for elucidating the signal pathway of sexual reproduction in V.virens.