ⅠGenetic Diversity And Pathogenicity Variation Of Different Rice Isolates In Sichuan Province ⅡCloning, Expression And Characterization Of G Proteinβ-subunit Gene In Rice Rhizoctonia Solani

Rice Rhizoctonia solani is deuteromycetes subphyl a fungi,a specie of Rhizoctonia,which has a wide host range and distribution.It cannot generate asexual spores and lives in soil by formation of sclerotia.R.solani,which is the pathogenic bacteria of field crops,such as Corn,paddy flee,lawn crops grass,potato.According to hyphal fusion classification,R.solani was divide into 14 fusion groups (AG-1—AG-13andAG-BI).In 1987,R.solani was divided into different intraspecific group(ISG)according to the cultivate characters,hosts,physiological,as well as biochemical.One of the fusion group AG-1 was then divided into three subgroups designated as AG-1ⅠA,AG-1ⅠB,AG-1ⅠC.The subgroup AG-1ⅠA of R.solani is the ascendant pathogenic flora of rice and corn sheath blight,which is the main diseaseoccurs for rice and corn and lead to a great loss of yield every year in the high temperature and high wet conditions of Yangtze River regions,Therefore,knowing the changing characteristics of Rice R.solani physiological race is very important to integrated control of rice sheath blight and resistancerice breeding to this disease.Heterotrimeric GTP-binding protein(G-protein)is composed byα,βand r subunits,which are implicated in major signal transduction pathways controlling a diversity of functions in the eukaryotic organisms,leading to the control of gene expression,cellular function and metabolism(Gilman 1987).In the inactive form it contains the GDP-boundα-subunit.Binding of ligand by the receptor causes exchange of GTP to GDP on theα-subunit.The G protein heterotrimer then dissociates intoα-GTP andβr-moieties.Depending on the system,either theα-GTP and theβr-unit can activate downstream cellular effectors.These effectors include cGMP phosphodiesterase,adenylycyclases,phospholipases and ion channels.In many phytopathogenic fungi such as Dictyostelium discoideum,Ustilagomaydis and Fusarium oxysporum,G protein are implicated in various biological processes,such as cell growth,differentiation and virulence.Research of Cryphonectria parasitica (Kasahara et al)showed that strain whose G proteinβ-subunit gene lost transgene function,whose hyphae was lack singnificant bifurcation and infection as well as pathogenicity decreased significantly.Fifty-five representative samples of rice R.solani were purified and analyzed for the pathogenicity and molecular genetic variation after they were collected and separated from five distinctive different ecological regions in Sichuan Province,the G proteinβ-subunit gene was isolated from rice R.solani by homologous cloning to do a preliminary study on its function,which laid a foundation for the studies on pathogenesis of rice R.solani and control methods.The main results were summarized as below:1.55 riceR.solani Stains were isolated by the agar without adding nutrition.2.The results of hyphal anastomosis test:all the isolated strains were fused with the standard strain,AG-1ⅠA,except the isolate D42.This cytological evidence also proved that the 54 strains of R.solani,which were fused with AG-11A,were belong to the same microbial population;Besides,some strains were also shown could be fused with more than one standard strains,as well as with many isolated strains.Therefore, these strains were suggested to be a category of R.solani named "bridge flora".3.Pathogenicity verification:The rice with X400,ShuHui527,ShuHui881 was selected in inoculation materials;leaves were collected and inoculated with the strains in vitro.The result showed that stains were significantly different in pathogenicity. Embedded inoculation method of Field showed the pathogenicity were significantly different with each other.At the same time,when inoculated different materials with the same stain in vitro showed little differences among the different materials,but in vivo which was opposite.4.RAPD analysis.According to the results of amplification,UPGMA molecule family tree of strains(isolated strains standard strains and outgroup control strain) were constructed by the software NNTSYS.All of the isolated strains except D42, D54 and AG-1ⅠA were classified into the same group when the similarity coefficient was 0.91,and the 9 international standard strains,AG-1ⅠB,AG-1ⅠC,AG-2-1, AG-2-2ⅢB,AG-2-2Ⅳ,AG-3,AG-4,AG-5,AG-6 and AG-BI,and the outgroup control strain A-1,were belong to different groups.respectively,all the 55 R.solani strains could be divided into 8 groups with the similarity coefficient was of 0.941.D1,D35,D6,D8,D30,D29,D48,D44,D9,D10,D26,D22,D12,D32,D28,D43,D49, D11,D24,D13,D14,D16,D15,D23,D21,D34,D38,D45,D4,D17,D51,D53,D18,D5,D31, D33,D52,D25,D27 and D19 were in group 1;D36,D39,D40,D46and D41were in group 2;D7and D20 which were collected from the south of Sichuan were in group 3; D37and D50 which from the east of Sichuan were in group 4;D2and D3 which from the southwest of Sichuan were in group 5;D47and D55 which from the west-east of Sichuan were in group 6;D42 from middle area of Sichuan was in group 7;D54 from the north,it was in the last group.5.Homologous cloning of G proteinβ-subunit gene:In the present study,isolation of theβ-subunit gene in R.solani AG-1ⅠA which causes rice sheath blight disease was reported.A 1960bp PCR fragment was obtained by utilizing of the specific primers degenerated from the conserved region in G proteinβ-subunit homologues from the Thanatephorus cucumeris.Amino acid sequence analysis showed that this sequence,a total of 366 deduced amino acid,encodes a putative peptide which shares significant identity with G proteinβ-subunit homologues from other organisms.6.G proteinβ-subunit gene polymorphism and phylogenetic analysis:The strain was identified as high homology according to the result of homology comparison of G proteinβ-subunit gene amino acid sequence,but there were also quite a few mutation in some interval.These sequences were obviously homologous with a lot of G proteinβ-subunit gene by phylogenetic analysis,whose similarity coefficients were from 88.14 to 57.34%7.Gene expression analysis:the expression of G proteinβ-subunit gene in R.solani AG-1ⅠA of different periods was mesured by semi-quantitive RT-PCR.The result showed there was no expression at the first 2 days,then increasing from the third to fifth days,which arrived maximum at the fifth day,And cut down to 0 after 6 days. So the amount of expression on the exponential phase of growth was the largest, which indicated that the expression of the G proteinβ-subunit gene in R.solani AG-1ⅠA might be regulated at transcription level.