Cloning And Expressing Of G-protein Beta-subunit In Rhizoctonia Solani Causing Rice Sheath Blight

Rice sheath blight caused by Rhizoctonia solani is one of three main diseases in rice all over the world.But recently with the expanding and aggravating degree of Rhizoctonia solani,the yield and quality of rice become more and more declinable,and it urgent for us to control rice sheath blight.Because Rhizoctonia solani is saprophytic fungus and has a extensive host range,it is difficult to use the traditional approach to find source of resistance.So far as no one has done deep research on resistant mechanism and genetics and breeding.Rice sheath blight was one of three serious rice diseases.The protein coding by Guanine nucleotide binding protein beta-subunit gene(G-protein beta-subunit) took an important role in pathopoiesis mechanism.In this paper,we aimed to identify and characterize the G-proteinβ-subunit from Rhizoctonia solani causing rice sheath blight. The genome of 1867bp and an open reading frame of 1047bp were amplified by PCR and RT-PCR.The genome of 1867bp(A-20.0%,C-26.6%,G-25.5%T-28.2%) included 4 introns and 5 exons.Introns ranged in size from 54 to 163bp,and their sequences complied with the rule of "5'-gt" and "ag-3'".The ORF predicted a 348-amino acid polypeptide with calculated molecular weight of 38.23 KDa and PI of 6.54.There were two alpha-helixes and seven beta sheets every of which included four beta-strands in amino acid secondary structure.In the tertiary structure two alpha-helixes in its N-terminal and seven beta sheets formed barrel structure by non-regular curl.The deduced amino acid sequence ofβ-subunit was 89%,88%,81%and 81%identical to the one from Lentinula edodes(AAT74567.1), Coprinopsis cinerea(EAU92269),Ustilago maydis(AAN33051) and Filobasidiella neoformans(AAD03596).The amplified OFR was cloned into the prokaryotic fusion expression vector pGEX-4T-2.E.coli BL21 was transformed with this recombinant construct and induced with IPTG for expression.The result indicated that the expressed protein size of ORF matched the prediction.