欢迎访问作物学报,今天是

作物学报 ›› 2011, Vol. 37 ›› Issue (02): 216-223.doi: 10.3724/SP.J.1006.2011.00216

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆丙二烯氧化物合酶基因(GmAOS)及其启动子的克隆与分析

吴娟娟1,2,吴倩1,喻德跃1,*   

  1. 1 南京农业大学国家大豆改良中心/作物遗传与种质创新国家重点实验室,江苏南京210095; 2 南通大学医学院生化教研室,江苏南通 226001
  • 收稿日期:2010-07-02 修回日期:2010-09-28 出版日期:2011-02-12 网络出版日期:2010-12-15
  • 作者简介:喻德跃, E-mail: dyyu@njau.edu.cn, Tel/Fax: 025-84396410
  • 基金资助:

    本研究由国家重点基础研究发展计划(973计划)项目(2010CB125906)资助。

Cloning and Characterization of GmAOS Gene and Its Promoter in Soybean (Glycine max)

WU Juan-Juan1,2,WU Qian1,YU De-Yue1,*   

  1. 1 National Center for Soybean Improvement/National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China; 2 Department of Biochemistry and Molecular Biology, Medical School of Nantong University, Nantong 226001, China
  • Received:2010-07-02 Revised:2010-09-28 Published:2011-02-12 Published online:2010-12-15
  • About author:喻德跃, E-mail: dyyu@njau.edu.cn, Tel/Fax: 025-84396410

摘要: 利用RT-PCR、RACE和LA PCR相结合的方法,从大豆中克隆了GmAOS基因及其启动子序列(登录号:EU366252),GmAOS基因共1 789 bp碱基,等电点8.97,分子量58.3 kD,在3种不同抗性大豆材料中均有2个拷贝。生物信息学分析表明,GmAOS酶的N末端有典型叶绿体定位信号肽,基因序列上有多个丝氨酸、苏氨酸、酪氨酸的磷酸化位点。该研究克隆到ATG上游472个碱基的GmAOS基因启动子部分序列,其含有赤霉素的响应元件(TAACAA),可诱导性抗性基因响应元件(W box),细菌和盐诱导的响应元件(GAAAAA),茉莉酸诱导的响应元件(G box)。GmAOS能强烈响应茉莉酸的诱导,且在黄皮小青豆(高抗斜纹夜蛾)中表达量高于徐疃大豆,两种材料抗虫性的差异可能是由GmAOS基因受诱导后的表达量差异引起的,即GmAOS基因与作物抗虫性相关,可做为培育高诱导抗性材料的候选基因。

关键词: 大豆, 丙二烯氧化物合酶, 启动子, 克隆, 生物信息学分析

Abstract: Allene oxide synthase (AOS) is a major intermediate enzyme in octadecanoid pathway to JA biosynthesis affecting the synthesis and levels of all JA-related compounds in plants, and therefore plays a signi?cant role in plant defense.In this study, a full length cDNA of GmAOS and its promoter were cloned from the soybean (Glycine max) by RT-PCR, RACE, and LA PCR methods. GmAOS cDNA coding 519 amino acids (58.3 kD) with an isoelectric point of 8.97 and two genes copies in the soybean genome coding for GmAOS. Bioinformatics analysis indicated that the N-terminal region of GmAOS displayed features of a typical chloroplast targeting peptide including an enrichment of serine, threonine and tyrosine phosphorylation sites. The length of the promoter was 472 bp, containing several stress-induced elements: GA inducing elements (TAACAA), W-box element which was in response to elicitor-responsive transcription of defense genes, element responsive to salt and pathogen (GAAAAA) and G-box (CACGTG) induced by JA. Jasmonic acid showed a strong inducement of the GmAOS transcript level, expression patterns of GmAOS were explored in two soybean accessions with distinct resistance to cotton worm: XTDD was highly susceptible and HPXQD highly resistant, showing that GmAOS had higher transcript level in HPXQD(HR) than in XTDD(HS). GmAOS transcript level were correlated with soybean material resistance grades. These results suggest GmAOS is likely to be a useful tool for improving self-resistance abality of high plants.

Key words: Soybean, A11ene oxide synthase, Promoter, Clone, Bioinformatics analysis

[1]Brash A R, Baertschi S W, Ingram C D, Harris T M. Isolation and characterization of natural allene oxides: unstable intermediates in tire metabolism of lipid hydriperoxides. Proc Natl Acad Sci USA, 1988, 85: 3382–3386
[2]Laudert D, Pfarmschmidt U, Lottspeich F, Holländer-Czytko H, Weiler E W. Cloning molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP74), the first enzyme of the octadecanoid pathway to jasmonatea. Plant Mol Biol, 1996, 31: 323–335
[3]Laudert D, Weiler E W. A11ene oxide symhase: a major control point in Arabidopsis thaliana octadecanoid signaling. Plant J, 1998, 15: 675–684
[4]Xu L(徐涛), Zhou Q(周强), Chen W(陈威). Effects of jasmonic acid signal transduction induced rice resistanceto to insect. Chin Sci Bull (科学通报), 2003, 48(13): 1442–1446 (in Chinese)
[5]Sivasankar S, Sheldrick B, Rothstein S J. Expression of allene oxide synthase determines defense gene activation in tomato. Plant Physiol, 2000, 122: 1335–1342
[6]Kongrit D, Jisaka M, Iwanaga C, Yokomichi H, Katsube T, Nishimura K, Nagaya T, Yokota K. Molecular cloning and functional expression of soybean allene oxide synthase. Biosci Biotechnol Biochem, 2007, 71: 491–498
[7]Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucl Acids Res, 1987, 15: 8125–8148
[8]Song W C, Funk C D, Brash A R. Molecular cloning of an allene oxide synthase: a cytochrome P450 specialized for the metabolism of fatty acid hydroperoxides. Proc Natl Acad Sci USA, 1993, 90: 8519–8523
[9]Maucher H, Hause B, Feussner I, Ziegler J, Wasternack C. Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seeding development. Plant J, 2000, 21: 199–213
[10]Howe G A, Lee G I, Itoh A, Li L, Derocher A E. Cytochrome P450-dependent metabolism of oxylipins in tomato: cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase. Plant Physiol, 2000, 123: 711–724
[11]Ziegler J, Keinänen M, Baldwin I T. Herbivore-induced allene oxide synthase transcripts and jasmonic acid in Nicotiana attenuate. Phytochemistry, 2001, 58: 729–738
[12]Song W C, Funk C D, Brash A R. Molecular cloning of an allene oxide synthase: a cytochrome P450 specialized for the metabolism of fatty acid hydroperoxides. Proc Natl Acad Sci USA, 1993, 90: 8519–8523
[13]Porter T D, Coon M J. Cytochrome P450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem, 1991, 266: 13469–13472
[14]Brash A R, Song W C. Structure-function features of flaxseed allene oxide synthase. Lipid Med Cell Signaling, 1995, 12: 275–282
[15]Farmer E E. Surfaee-to-air signals. Nature, 2001, 411: 854–856
[16]Ariumra G, Ozawa R. Herbivory induced volatiles elicit defence genes in lima bean leaves. Nature, 2000, 406: 512–514
[17]Wu J J, Wu Q, Wu Q J, Gai J Y, Yu D Y. Constitutive overexpression of AOS-like gene from soybean enhanced tolerance to insect attack in transgenic tobacco. Biotechnol Lett, 2008, 30: 1693–1698
[18]Berger S. Jasmonate-related mutants of Arabidopsis as tools for studying stress signaling. Planta, 2001, 214: 497–504
[1] 崔连花, 詹为民, 杨陆浩, 王少瓷, 马文奇, 姜良良, 张艳培, 杨建平, 杨青华. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
[2] 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.
[3] 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.
[4] 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.
[5] 李海芬, 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强. 花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析[J]. 作物学报, 2022, 48(6): 1558-1565.
[6] 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.
[7] 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.
[8] 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.
[9] 周慧文, 丘立杭, 黄杏, 李强, 陈荣发, 范业赓, 罗含敏, 闫海锋, 翁梦苓, 周忠凤, 吴建明. 甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析[J]. 作物学报, 2022, 48(4): 1017-1026.
[10] 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.
[11] 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.
[12] 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.
[13] 徐宁坤, 李冰, 陈晓艳, 魏亚康, 刘子龙, 薛永康, 陈洪宇, 王桂凤. 一个新的玉米Bt2基因突变体的遗传分析和分子鉴定[J]. 作物学报, 2022, 48(3): 572-579.
[14] 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.
[15] 杨昕, 林文忠, 陈思远, 杜振国, 林杰, 祁建民, 方平平, 陶爱芬, 张立武. 黄麻双生病毒CoYVV的分子鉴定和抗性种质筛选[J]. 作物学报, 2022, 48(3): 624-634.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!