立枯丝核菌（水稻纹枯病菌）G蛋白β亚基基因的克隆与特性分析

Cloning and expression of G-protein beta-subunit in rice Rhizoctonia solani

The protein coding by G-protein beta-subunit plays an important role in pathogenesis mechanism. In this paper,the G-protein β subunit from Rhizoctonia solani causing rice sheath blight was identified. The genome of 1867bp and an open reading frame (ORF) of 1047bp were amplified by PCR and RT-PCR. The genome included 4 introns and 5 exons. Introns ranged in size from 54 to 65bp,and its sequence complied with the rule of " 5′-gt " and " ag-3′ ". The ORF predicted a 348-amino acid polypeptide with calculated molecular weight of 38.23kDa and PI of 6.54. There were two alpha-helixes and seven beta sheets including four beta-strands each 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 of β-subunit was 89%,88%,81% and 81%,being identical to that from Lentinula edodes (AAT74567.1),Coprinopsis cinerea (EAU92269),Ustilago maydis (AAN33051) and Filobasidiella neoformans (AAD03596). The amplified ORF was cloned into the prokaryotic fusion expression vector pGEX-4T-2. E. coli BL21 was transformed by this recombinant construct and induced by Isopropyl β-D-1-Thiogalactopyranoside (IPTG) for expression. The result indicated that the expressed protein size of ORF matched the prediction.