The Research On The Interacting Proteins With Transcription Factor SsMCM1 In Sclerotinia Sclerotiorum

(Sclerotinia sclerotiorum?Lib?de Bary)is a typical type of necrotrophic plant pathogenic fungi with a very broad range of hosts,can infect at least 75 families 278 genera and 408 kinds of plants,including important crops,fruits and vegetables such as soybeans,beans,green beans,sunflower,rape,tomato,green pepper,lettuce and many other plants,causing serious economic losses.The MADS-box family is an important transcriptional regulator in eukaryotes and is found in plants,insects,nematodes,fungi,low vertebrates and mammals.It regulates a variety of cellular functions including primary metabolism,cell cycle,cell identification and so on.We cloned SsMCM1 of S.sclerotiorum,which indicated that SsMCM1 had a highly conserved DNA binding junction,the domains belonged to SRF–like MADS-box family.Then gene silencing study showed that SsMCM1 was involved in the regulation of the growth,pathogenicity and sexual reproduction of mycelium of S.sclerotiorum.Base on this work,in order to clarify the SsMCM1 transcription factor regulation mechanism and control network we carried out the following research content:1.pGBKT7-SsMCM1 vector was constructed and introduced into yeast cells for toxicity and self-activation.The results showed that pGBKT7-SsMCM1 vector was non-Toxic to yeast cells and had no self-activating activity.Screen the cDNA library of S.sclerotiorum by yeast two-hybrid technique and confirm the results by co-transfection.We selected 13 proteins with possible interactions and numbered as follows: Ss IP31,SsIP34,SsIP37,SsIP38,SsIP43,SsIP44,SsIP63,SsIP80,SsIP86,SsIP98,SsIP99,SsIP100 and SsIP106.2.After 13 samples sequenced,the primers were designed for cloning the full-length gene which was ligated to the pGADT7 vector and co-transgenic with the pGBKT7-SsMCM1 vector and identify 12 interacting proteins.3.To compare the cell pathways in yeast,biosynthesis analysis of SsMCM1 found out that there was a protein SSIG₀2296 which was highly homologous to Cdc28,downstream of yeast MCM1 transcription factor.The gene was cloned and named SsCdc28.Then we constructed the pGBKT7-SsCdc28 vector,and screened a protein SsIP86 which interacts with both SsCdc28 and SsMCM1 through yeast two-hybrid.4.In order to further verify the interaction between SsMCM1-SsIP86 and SsIP86-SsCdc28 proteins,bimolecular fluorescent complementary vectors were constructed and the protoplast transformation of Arabidopsis thaliana was carried out.The fluorescence was observed by confocal microscopy.The results showed that SsMCM1 could interacte with SsIP86,and interaction is carried out in the nucleus.In this study,12 proteins interacting with SsMCM1 transcription factor were screened by yeast two-hybridization.SsMCM1 could interacte with SsIP86,and interaction is carried out in the nucleus.Yeast two-hybrid showed that SsMCM1 interacted with SsIP86,SsIP86 interacted with SsCdc28 These studies provide new insights into the regulatory mechanism of transcription factor SsMCM1.