欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2009, Vol. 35 ›› Issue (2): 370-374.doi: 10.3724/SP.J.1006.2009.00370

• 研究简报 • 上一篇    下一篇

大豆立枯丝核菌G蛋白ß亚基基因的克隆与分析

马炳田1,2;曲广林1;黄文娟1;林瑜凡1;李仕贵1,2,*   

  1. 1四川农业大学水稻研究所，成都温江611130；2四川农业大学/西南作物基因资源与遗传改良教育部重点实验室，四川雅安625014
  • 收稿日期:2008-05-18 修回日期:2008-09-10 出版日期:2009-02-12 网络出版日期:2008-12-12
  • 通讯作者: 李仕贵
  • 基金资助:

    本研究由教育部长江学者和创新团队发展计划项目（IRT0453）资助。

Cloning and Analyzing of G-protein Beta-Subunit Gene in Rhizoctonia solani Causing Soybean Sharp Eyespot

MA Bing-Tian1,2,QU Guang-Lin1,HUANG Wen-Juan1,LIN Yu-Fan1,LI Shi-Gui1,2,*   

  1. 1Rice Research Institute, Sichuan Agricultural University, Chengdu 611130,China;2 Key Laboratory of Crop Genetic Resoures and Improvement, Ministry of Education, Sichuan Agricultural University,Ya'an 625014,China
  • Received:2008-05-18 Revised:2008-09-10 Published:2009-02-12 Published online:2008-12-12
  • Contact: LI Shi-Gui

PDF

1268

被引次数

1

摘要：

由立枯丝核菌[Rhizoctonia solani Kühn，有性世代：Thanatephorus cucumeris (Frank) Donk] 引起的大豆纹枯病(Soybean sharp eyespot)是一种重要病害。G蛋白β亚基(Guanine nucleotide binding protein beta-subunit)作为重要的信号传导因子，在植物病原菌致病分子机制中起着重要作用。为了解G蛋白β亚基基因的结构与功能，根据同源物种G蛋白β亚基相关序列设计引物，利用PCR和RT-PCR技术克隆了大豆立枯丝核菌G蛋白β亚基的基因序列和开放阅读框(G-protein beta-subunit of Soybean Rhizoctonia solani，简写为gbsrs1，GenBank登录号为EU663628)。该片段全长1 864 bp，含有4个内含子和5个外显子；开放阅读框(ORF)长1 047 bp，编码348氨基酸残基，与多种真菌G蛋白β亚基的氨基酸序列相似程度较高，达79.72%~99.43%；该蛋白质具有2个α-螺旋和7个β-折叠的二级结构，形成无规则卷曲连接的桶形三级结构。将gbsrs1的ORF连接于原核融合表达载体pGEX-4T-2中，经IPTG诱导，获得了相应蛋白的表达。gbsrs1的克隆和特性研究为了解大豆立枯丝核菌的致病机理、有效防治纹枯病奠定了基础。

关键词: 大豆, 立枯丝核菌, 致病性, G蛋白ß亚基, 信号传导

Abstract:

Soybean sharp eyespotis one of the most serious diseases in world. The protein encoded by G-protein β-subunit (Guanine nucleotide binding protein beta-subunit) gene plays an important role in pathogenesis mechanism. In this paper, the G-protein β-subunit from Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris)causing soybean sharp eyespot was identified. The genome of 1 864 and 1 047 bp open reading frame (ORF) were amplified by PCR and RT-PCR. The gene included 4 introns and 5 exons. Introns ranged in size from 54 to 65 bp, and their sequences complied with the rule of “5'-gt” and “ag-3'” (GenBank Accession No. EU663628). The ORF encoded 348-amino acid polypeptide with 38.24 kD of calculated molecular weight and 6.31 of pI. There were two alpha-helixes and seven beta-sheets including four beta-strands each in its amino acid secondary structure. Two alpha-helixes in its N-terminal and seven beta-sheets formed barrel structure by non-regular curl in the tertiary structure. The deduced amino acid sequence of β-subunit was identical to that from Rhizoctonia solani (GenBank Accession No. EU267677, AY884129), Lentinula edodes (GenBank Accession No. AAT74567), Coprinopsis cinerea (GenBank Accession Number EAU92269), Ustilago maydis (GenBank Accession Number AAN33051) and Filobasidiella neoformans (GenBank Accession No. AAD03596) with 99.43%, 89.19%, 87.97%, 83.66%, 80.23%, and 79.72%, respectively. The amplified ORF was ligated into the prokaryotic fusion expression vector pGEX-4T-2. E. coli BL21 was transformed with this recombinant vector and induced by IPTG for expression. The result indicated that the protein size of ORF matched the prediction. This cloning of this gene provides the evidence for controlling hyphal growth, development and virulence in R. solani.

Key words: Soybean, Rhizoctonia solani, Pathology, G-protein beta-subunit, Signal transduction

[1]Zhai Z-H(翟中和), Wang X-Z(王喜忠), Ding M-X(丁明孝). Cell Biology (细胞生物学). Beijing: Higher Education Press, 2000. pp 124–157(in Chinese)
[2]Ford C E, Skiba N P, Bae H, Daaka Y, Reuveny E, Shekter L R, Rosal R, Weng G, Yang C S, Iyengar R, Miller R J, Jan L Y, Lefkowitz R J, Hamm H E. Molecular basis for interations of G protein βγ subunits with effectors. Science, 1998, 280: 1271–1274
[3]Chen J-L(陈巨莲), Ni H-X(倪汉祥), Sun J-R(孙京瑞), Weng G. G protein β1γ2 subunits purification and their interaction with adenylyl cyclase. Sci China (Ser C) (中国科学?C辑), 2003, 33(1): 56–64 (in Chinese)
[4]Hou Y M, Chang V, Capper A B, Taussig R, Gautam N. G protein β subunit types differentially interact with a muscarinic receptor but not adenylyl cyclase type II or phospholipase C-β2/3. J Biol Chem, 2001, 276: 19982–19988
[5]Kasahara S, Nuss D L. Targeted disruption of a fungal G-protein β subunit gene results in increased vegetative growth but reduced viru-lence. Mol Plant Microbe Int, 1997, 10: 984–993
[6]Latijnhouwers M, Govers F. A Phytophthora infestans G-Protein β subunit is involved in sporangium formation. Eukaryot Cell, 2003, 2: 971–977
[7]Zeller C E, Parnell S C, Dohlman H G. The RACK1 ortholog Asc1 functions as a G-protein β-Subunit coupled to glucose responsiveness in yeast. J Biol Chem, 2007, 282: 25168–25176
[8]Delgado-Jarana J, Martínez-Rocha A L, Roldán-Rodriguez R, Ron-cero M I, Di Pietro A. Fusarium oxysporum G-protein beta subunit Fgb1 regulates hyphal growth, development, and virulence through multiple signalling pathways. Fungal Genet Biol, 2005, 42: 61–72
[9]Chen J-L(陈巨莲), Weng G-Z, Ni H-X(倪汉祥). The advancement of G protein and coupled signal transduction pathways. Chin J Biotech-nol (生物工程学报), 2001, 17(2): 113–117(in Chinese with English abstract)
[10]Ruiz-Velasco V, Ikeda S R, Puhl H L. Cloning, tissue distribution and functional expression of the human G protein β4-subunit. Physiol Genomics, 2002, 8: 41–50
[11]Lupas A N, Lupas J M, Stock J B. Do G protein subunits associate via a three-stranded coiled coil? FEBS Lett, 1992, 314: 105–108
[12]Claphan D E, Neer E J. New roles for G-protein βγ-dimers in trans-membrane signaling. Nature, 1993, 365: 403–406
[13]Wang D S, Shaw R, Winkelmann J C, Shaw G. Binding of PH do-mains of β-adrenergic receptor kinase and β-spectrin to WD40/ β-transducin repeat containing regions of the β-subunit of trimeric G-proteins. Biochem Biophys Res Commun, 1994, 203: 29–35
[14]Weiss C A, Garnaat C W, Mukai K, Hu Y, Ma H. Isolation of cDNAs encoding GTP-binding protein β-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci USA, 1994, 91: 9554–9558
[15]Ishikawa A, Iwasaki Y, Asahi T. Molecular cloning and characteriza-tion of a cDNA for the β-subunit of a G protein from rice. Plant Cell Physiol, 1996, 37: 223–228
[16]Kaydamov C, Tewes A, Adler K, Manteuffel R. Molecular charac-terization of cDNAs encoding G protein α and β subunits and study of their temporal and spatial expression patterns in Nicotiana plum-baginifolia Viv. Biochim Biophys Acta, 2000, 149: 143–160
[17]Wang P, Perfect J R, Heitman J. The G-protein β subunit GPB1 is re-quired for mating and haploid fruiting in Cryptococcus neoformans. Mol Cell Biol, 2000, 20: 352–362
[1] 陈玲玲, 李战, 刘亭萱, 谷勇哲, 宋健, 王俊, 邱丽娟. 基于783份大豆种质资源的叶柄夹角全基因组关联分析[J]. 作物学报, 2022, 48(6): 1333-1345.
[2] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[3] 王炫栋, 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花. 拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究[J]. 作物学报, 2022, 48(6): 1546-1557.
[4] 于春淼, 张勇, 王好让, 杨兴勇, 董全中, 薛红, 张明明, 李微微, 王磊, 胡凯凤, 谷勇哲, 邱丽娟. 栽培大豆×半野生大豆高密度遗传图谱构建及株高QTL定位[J]. 作物学报, 2022, 48(5): 1091-1102.
[5] 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[6] 彭西红, 陈平, 杜青, 杨雪丽, 任俊波, 郑本川, 罗凯, 谢琛, 雷鹿, 雍太文, 杨文钰. 减量施氮对带状套作大豆土壤通气环境及结瘤固氮的影响[J]. 作物学报, 2022, 48(5): 1199-1209.
[7] 王好让, 张勇, 于春淼, 董全中, 李微微, 胡凯凤, 张明明, 薛红, 杨梦平, 宋继玲, 王磊, 杨兴勇, 邱丽娟. 大豆突变体ygl2黄绿叶基因的精细定位[J]. 作物学报, 2022, 48(4): 791-800.
[8] 李瑞东, 尹阳阳, 宋雯雯, 武婷婷, 孙石, 韩天富, 徐彩龙, 吴存祥, 胡水秀. 增密对不同分枝类型大豆品种同化物积累和产量的影响[J]. 作物学报, 2022, 48(4): 942-951.
[9] 杜浩, 程玉汉, 李泰, 侯智红, 黎永力, 南海洋, 董利东, 刘宝辉, 程群. 利用Ln位点进行分子设计提高大豆单荚粒数[J]. 作物学报, 2022, 48(3): 565-571.
[10] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[11] 王娟, 张彦威, 焦铸锦, 刘盼盼, 常玮. 利用PyBSASeq算法挖掘大豆百粒重相关位点与候选基因[J]. 作物学报, 2022, 48(3): 635-643.
[12] 董衍坤, 黄定全, 高震, 陈栩. 大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能[J]. 作物学报, 2022, 48(2): 353-366.
[13] 张国伟, 李凯, 李思嘉, 王晓婧, 杨长琴, 刘瑞显. 减库对大豆叶片碳代谢的影响[J]. 作物学报, 2022, 48(2): 529-537.
[14] 禹桃兵, 石琪晗, 年海, 连腾祥. 涝害对不同大豆品种根际微生物群落结构特征的影响[J]. 作物学报, 2021, 47(9): 1690-1702.
[15] 宋丽君, 聂晓玉, 何磊磊, 蒯婕, 杨华, 郭安国, 黄俊生, 傅廷栋, 汪波, 周广生. 饲用大豆品种耐荫性鉴定指标筛选及综合评价[J]. 作物学报, 2021, 47(9): 1741-1752.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2