| 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 |
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| 中文关键词:信号传导，开放阅读框，原核表达 |
| 英文关键词:signal transduction, ORF, prokaryotic fusion expression |
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| 中文摘要: |
| 由立枯丝核菌Rhizoctonia solani引起的水稻纹枯病（rice sheath blight）是水稻三大病害之一。G蛋白β亚基（G-protein beta-subunit）编码的蛋白作为重要的信号传导蛋白，在其致病分子机制中起着重要作用。为了解G蛋白β亚基基因的作用方式，本文根据同源物种G蛋白β亚基相关序列设计引物，通过PCR（polymerase chain reaction）和RT-PCR（reverse transcriptase PCR）技术，获得了以水稻为寄主的立枯丝核菌G蛋白β亚基（G-protein beta-subunit of rice Rhizoctonia solani，简写gbrrs1）的基因序列和开放阅读框ORF（open reading frame）（GenBank 登录号EU267677）。该基因全长1867bp，含有4个内含子和5个外显子，各内含子长度在54bp-65bp，且序列均符合5′-gt……ag-3′模式。开放阅读框1047bp，编码348aa，推测的蛋白质分子量为38.23kDa，等电点为6.54。该蛋白质具有2个alpha-helix和7个beta sheet的二级结构，每个beta sheet又包含4个beta-strand。gbrrs1在Ｎ端有2个alpha-helix，紧接着是由7个beta sheet由无规则卷曲连接形成的桶形结构。GenBank Blast结果表明，gbrrs1与四种真菌生物G蛋白β亚基的氨基酸序列同源性较高，与Lentinula edodes（AAT74567.1）、Coprinopsis cinerea（EAU92269）、Ustilago maydis（AAN33051）和Filobasidiella neoformans（AAD03596）的一致性分别达到了89%、88%、81%和81%。将gbrss1的开放阅读框克隆于原核融合表达载体pGEX-4T-2中，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，获得了相应蛋白的表达。 |
| 英文摘要: |
| 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. |
| 曲广林 李仕贵 徐正君 王玉平 黄文娟 林瑜凡 万佳 马炳田. 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[J]. 菌物学报, 2008, 27(5): 718-726 |
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