欢迎访问作物学报,今天是

作物学报 ›› 2015, Vol. 41 ›› Issue (12): 1819-1827.doi: 10.3724/SP.J.1006.2015.01819

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

向日葵病程相关蛋白HaPR1基因的克隆与功能研究

马立功1,2,张匀华2,*,孟庆林2,石凤梅2,刘佳2,李易初2,王志英1,*   

  1. 1东北林业大学林学院,黑龙江哈尔滨,150040;2黑龙江省农业科学院植物保护研究所,黑龙江哈尔滨,150086
  • 收稿日期:2015-04-20 修回日期:2015-07-20 出版日期:2015-12-12 网络出版日期:2015-08-11
  • 通讯作者: 王志英, E-mail: zyw0451@sohu.com, Tel: 13069873855; 张匀华, E-mail: yhzhang9603@126.com, Tel: 13603639603
  • 基金资助:

    本研究由国家现代农业产业技术体系建设专项(CARS-16)和黑龙江省农业科技创新工程项目(QN015)资助。

Cloning and Function Analysis of Pathogenesis Related Protein Gene HaPR1 from Sunflower (Helianthus annuus)

MA Li-Gong1,2,ZHANG Yun-Hua2,*,MENG Qing-Lin2,SHI Feng-Mei2,LIU Jia2,LI Yi-Chu2,WANG Zhi-Ying1,*   

  1. 1 College of Forestry, Northeast Forestry University, Harbin 150040, China; 2 Institute of Plant Protection, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
  • Received:2015-04-20 Revised:2015-07-20 Published:2015-12-12 Published online:2015-08-11
  • Contact: 王志英, E-mail: zyw0451@sohu.com, Tel: 13069873855; 张匀华, E-mail: yhzhang9603@126.com, Tel: 13603639603
  • Supported by:

    This research was supported by the Modern Agro-industry Technology Research System (CARS-16) and the Agricultural Science and Technology Innovation Program of Heilongjiang Province (QN015).

摘要:

病程相关蛋白(pathogenesis-related proteins)经常被用作植物抗病反应的分子标记。本文在核盘菌诱导向日葵转录组文库的基础上克隆1个病程相关蛋白1基因HaPR1的cDNA全长序列,并进行了表达模式和功能分析。结果表明, 该基因cDNA全长开放阅读框为489 bp,编码162个氨基酸,分子量为17.52 kD,等电点为8.19,具有6个保守半胱氨酸,4个保守的allergen V5/Tpx-1结构域,GenBank登录号为KR071874。经比较HaPR1与多种物种PR1高度同源。实时荧光定量PCR检测结果表明,HaPR1相对表达量在向日葵叶中最高,根中其次,茎中最低。干旱、盐、草酸、核盘菌及其代谢物均可显著诱导其表达。利用农杆菌介导法将该基因导入烟草,提高了转基因株系对核盘菌的抗性。对抗性株系烟草防御酶活性及丙二醛含量测定发现转基因烟草叶片在核盘菌胁迫下显著提高了SOD、POD和CAT活性,降低了MDA含量。初步推断HaPR1具有抗核盘菌的功能。

关键词: 向日葵, 病程相关蛋白1, 基因克隆, 功能分析

Abstract:

Pathogenesis-related proteins are commonly used as markers of plant defense responses. The full-length cDNA of pathogenesis-related protein 1 (PR1) named HaPR1 in Helianthus annuus was cloned based on the transcriptome of H.annuus induced by Sclerotinia sclerotiorum, and its expression model and function were analyzed in this study. Sequence analysis showed that the cDNA of HaPR1 (GenBank No. KR071874) contained a 489 bp ORF encoding a protein of 162 amino acids residues with the molecular mass of 17.52 kD and theoretical pI of 8.19, HaPR1 possessed six conserved cysteine and four conserved allergen V5/Tpx-1 related domain. The HaPR1 was highly homologous with PR1 in other species. Real-time PCR analysis showed that expression level of HaPR1 was the highest in leaf, and was significantly induced by drought, salt stress, oxalic acid, S. sclerotiorum and its metabolites. Then the HaPR1 gene was transformed into tobacco by Agrobacterium tumefaciens to further verify its function. The results showed that the expression of HaPR1 improved the resistance of transgenic lines, and significantly increased SOD, POD, and CAT activities and reduced the content of MDA. It suggested that HaPR1 has a function of resistance to S. sclerotiorum.

Key words: Helianthus annuus, Pathogenesis-related protein 1, Gene clone, Function analysis

[1]谢纯政, 刘海燕, 李玲, 梁炫强. 植物病程相关蛋白PR10研究进展. 分子植物育种, 2008, 6: 949–953



   Xie C Z, Liu H Y, Li L, Liang X Q. Advances on class 10 pathogenesis-related proteins. Mol Plant Breed, 2008, 6: 949–953 (in Chinese with English abstract)



[2]Loon L C, Strien E A. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol, 1999, 55: 85–97



[3]Van Loon L C, Rep M, Pieterse C M J. Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol, 2006, 44: 135–162



[4]牛吉山, 刘瑞, 郑磊. 小麦PR-1、PR-2、PR-5基因的白粉菌和水杨酸诱导表达分析及白粉病抗性研究. 麦类作物学报, 2007, 27: 1132–1137



    Niu J S, Liu R, Zheng L. Expression analysis of wheat PR-1, PR-2, PR-5 activated by Bgt and SA, and powdery mildew resistance. J Triticeae Crops, 2007, 27: 1132–1137 (in Chinese with English abstract)



[5]Xie X Z, Xue Y J, Zhou J J, Zhang B, Chang H, Takano M. Phytochromes regulate SA and JA signaling pathways in rice and are required for developmentally controlled resistance to Magnaporthe grisea. Mol Plant, 2011, 4: 688–696



[6]Nandi A, Kachroo P, Fukushige H, Hildebrand D F, Klessig D F, ShahJ. Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant. Mol Plant Microbe Interact, 2003, 16: 588–599



[7]Flors V, Ton J, van Doorn R, Jakab G, García-Agustín P, Mauch-Mani B. Interplay between JA, SA and ABA signalling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicicola. Plant J, 2008, 54: 81–92



[8]Alexander D, Goodman R M, GutRella M, Glascock C, Weymann K, Friedrich L, Maddox D, Ahlgoy P, Luntz T, Ward E. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc Natl Acad Sci USA, 1993, 90: 7327–7331



[9]NiKi T, Mitsuhara I, Seo S. Antagonistic effect of Salicylic 330 acid and jasmonic acid on the expression of pathogeneis-relation (PR) protein genes in wounded mature tobacco leaves. Plant Cell Physiol, 1998, 39: 500–507



[10]Sarowar S, Kim Y J, Kim E N. Overexpression of a pepper basic pathogenesis-related protein1 gene in tobacco plants enhances resistance to heavy metal and pathogen stresses. Plant Cell Rep, 2005, 24: 216–224



[11]Li Z T, Dhekney S A, Gray D J. PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco. Plant Cell Rep, 2011, 30: 1–11



[12]Bonasera J M, Kim J F, Beer S V. PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora. BMC Plant Biol, 2006, 6: 23



[13]Seo P J, Lee A K, Xiang F N, Park C M. Molecular and functional profiling of Arabidopsis pathogenesis-related genes: insights into their roles in salt response of seed germination. Plant Cell Physiol, 2008, 49, 334–344



[14]Stintzi A, Heitz T, Kauffmann S, Legrand M, Fritig B. Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco as osmotin. Physiol Mol Plant Pathol, 1991, 38: 137–146



[15]Agrawal G K, Jwa N S, Rakwal R. A novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors. Biochem Biophys Res Commun, 2000, 274: 157–165



[16]Agrawal G K, Rakwal R, Jwa N S. Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals. Biochem Biophys Res Commun, 2000, 278: 290–298



[17]Agrawal G K, Rakwal R, Jwa N S, Agrawal V P. Signaling molecules and blast pathogen attack activates rice OsPR1a and OsPR1b genes: a model illustrating components participating during defence/stress response. Plant Physiol Biochem, 2001, 39: 1095–1103



[18]Livak K, Schmittgen, T. Analysis of relative gene expression data using real-time quantitative PCR and the 2-delta delta CT method. Methods, 2001, 25: 402–408



[19]邹琦. 植物生理生化实验指导. 北京: 农业出版社, 1991



    Zou Q. Plant Physiological and Biochemical Experiments. Beijing: Agriculture Press, 1991 (in Chinese)



[20]朱广廉, 钟文海, 张爱琴. 植物生理实验. 北京: 北京大学出版社, 1991



    Zhu G L, Zhong W H, Zhang A Q. Plant Physiology Experiment. Beijing: Peking University Press, 1991 (in Chinese)



[21]汤章城, 王国强, 史益敏. 现代植物生理学实验指南. 北京: 科学出版社, 1999



    Tang Z C, Wang G Q, Shi Y M. Modern Experiment Guide for Plant Physiology. Beijing: Science Press, 1999 (in Chinese)



[22]陈建勋, 王晓峰. 植物生理学实验指导. 广州: 华南理工大学出版社, 2002. pp 31–32, 117–120



    Chen J X, Wang X F. Experiment Manual for Plant Physiology. Guangzhou: South China University of Technology Press, 2002. pp 31–32, 117–120 (in Chinese)



[23]Kim S T, Yu S, Kang Y H. The rice pathogen-related protein 10 (Jiospr10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity. Plant Cell Rep, 2008, 27: 593–603



[24]张计育, 渠慎春, 薛华柏, 高志红, 郭忠仁, 章镇. 湖北海棠病程相关蛋白MhPR8基因的克隆与表达. 中国农业科学, 2012, 45: 1568–1575



    Zhang J Y, Qu S C, Xue H B, Gao Z H, Guo Z R, Zhang Z. Isolation and expression of pathogenesis-related protein gene MhPR8 from Malus hupehensis. Sci Agric Sin, 2012, 45: 1568–1575 (in Chinese with English abstract)



[25]栗小英, 高琳, 张艳俊, 王海燕, 刘大群. 叶锈菌及信号分子诱导小麦TcLr35中β-1,3-葡聚糖酶基因的表达分析. 中国农业科学, 2014, 47: 2774–2783



    Li X Y, Gao L, Zhang Y J, Wang H Y, Liu D Q. Expression and analysis of β-1,3-glucanase gene in wheat TcLr35 induced by wheat leaf rust pathogen and signal molecule. Sci Agri Sin, 2014, 47: 2774–2783 (in Chinese with English abstract)



[26]侯丽霞, 高超, 车永梅, 赵方贵, 刘新. 葡萄病程相关蛋白1基因的克隆和表达分析. 植物生理学报, 2012, 48: 57–62



    Hou L X, Gao C, Che Y M. Gene cloning and expression analysis of pathogenesis-related protein 1 in Vitis vinifera L. Plant Physiol J, 2012, 48: 57–62 (in Chinese with English abstract)



[27]王艳, 陈西, 周莲洁, 杨中敏. 费尔干猪毛菜病程相关蛋白基因SfPR-1的表达规律和植物表达载体构建. 生物技术通报, 2014, 1: 117–124



    Wang Y, Chen X, Zhou L J, Yang Z M. Expression profiles of pathogen-related protein gene (SfPR-1) from Salsola ferganica and construction of plant expression vectors. Biotechnol Bull, 2014, 1: 117–124 (in Chinese with English abstract)

[1] 左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[2] 宋裕祯, Bheel Chander Kumar, 王跃, 张颖星, 郭娟, Khound Rituraj, Santra Dipak Kumar, 曹晓宁, 王瑞云. 糜子AP2亚家族全基因组鉴定及PmAP2-1PmAP2-9耐盐功能分析[J]. 作物学报, 2026, 52(4): 1127-1139.
[3] 汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[4] 张晴, 杨昱, 郭茜, 岳霈尧, 殷丛丛, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 大豆GmARA6a的克隆及响应盐胁迫的功能分析[J]. 作物学报, 2026, 52(2): 480-493.
[5] 余开航, 周洪斌, 罗亮扎, 王玫郦, 姜瑞梅, 董陈文华, 李仕金, 毛孝强, 陈升位. 大麦亮氨酸富集重复型类受体激酶基因HvLRR-RLK-510的克隆和表达分析[J]. 作物学报, 2026, 52(2): 421-432.
[6] 薛晓菲, 戴云静, 李熙林, 丁艳艳, 王翔, 雷长英, 韩焕勇, 贺道华. 陆地棉杜松烯合酶基因GhCDN10的特征及其在棉酚合成中功能分析[J]. 作物学报, 2025, 51(8): 2060-2076.
[7] 王林, 陈晓雨, 张文梦龙, 汪思琦, 程冰云, 程靖秋, 潘锐, 张文英. 大麦HvMYB2分子特性及响应干旱胁迫的功能分析[J]. 作物学报, 2025, 51(4): 873-887.
[8] 张正康, 苏延红, 阮孙美, 张敏, 张攀, 张慧, 曾千春, 罗琼. 疣粒野生稻中OgXa13的克隆和功能研究[J]. 作物学报, 2025, 51(2): 334-346.
[9] 王玉娇, 王永乐, 添长久, 郁春旺, 吕佳斌, 朱加保. 薏苡VQ4基因的克隆及耐盐性初步分析[J]. 作物学报, 2025, 51(12): 3198-3210.
[10] 张顺杰, 吴维泰, 冉禧玥, 赵梓含, 韩永辉, 吴正丹, 张凯. 甘薯β淀粉酶基因IbBAM48829的功能解析[J]. 作物学报, 2025, 51(11): 3096-3104.
[11] 李旭娟, 李纯佳, 田春艳, 孔春艳, 徐超华, 刘新龙. 甘蔗硝酸盐转运蛋白1/肽转运蛋白家族6.4基因(ScNPF6.4)克隆及其调控分蘖功能分析[J]. 作物学报, 2024, 50(8): 2131-2142.
[12] 李闻娟, 王利民, 齐燕妮, 赵玮, 谢亚萍, 党照, 赵丽蓉, 李雯, 徐晨梦, 王琰, 张建平. 亚麻LuWRI1a在旱盐胁迫响应中的功能分析[J]. 作物学报, 2024, 50(7): 1750-1761.
[13] 李海芬, 鲁清, 刘浩, 温世杰, 王润风, 黄璐, 陈小平, 洪彦彬, 梁炫强. 花生赤霉素3-β-双加氧酶(AhGA3ox)基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2024, 50(4): 932-943.
[14] 张宝华, 刘佳静, 田晓, 田旭钊, 董阔, 武郁洁, 肖凯, 李小娟. 小麦TaSPX1基因的克隆、表达及耐低氮逆境的功能研究[J]. 作物学报, 2024, 50(3): 576-589.
[15] 杨晨曦, 周文期, 周香艳, 刘忠祥, 周玉乾, 刘芥杉, 杨彦忠, 何海军, 王晓娟, 连晓荣, 李永生. 控制玉米株高基因PHR1的基因克隆[J]. 作物学报, 2024, 50(1): 55-66.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!