欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2019, Vol. 45 ›› Issue (8): 1158-1165.doi: 10.3724/SP.J.1006.2019.81017

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦类受体蛋白激酶基因TaPK3A的克隆与抗纹枯病功能初步分析

苏强,荣玮,张增艳()   

  1. 中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与遗传育种重点实验室, 北京 100081
  • 收稿日期:2018-09-30 接受日期:2019-01-19 出版日期:2019-08-12 网络出版日期:2019-07-16
  • 通讯作者: 张增艳
  • 作者简介:E-mail: 243939427@qq.com
  • 基金资助:
    本研究由国家自然科学基金项目资助(31771789)

Cloning and functional analysis of wheat receptor-like kinase gene TaPK3A

SU Qiang,RONG Wei,ZHANG Zeng-Yan()   

  1. National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-09-30 Accepted:2019-01-19 Published:2019-08-12 Published online:2019-07-16
  • Contact: Zeng-Yan ZHANG
  • Supported by:
    This study was support by the National Natural Science Foundation of China(31771789)

RichHTML

11

PDF

838

摘要：

小麦纹枯病已成为我国小麦生产的重要限制因素。研究小麦防御反应分子基础, 发掘有效的抗病基因是小麦抗纹枯病育种突破的前提。本研究从抗纹枯病小麦品系CI12633中克隆出一个类受体蛋白激酶(receptor-like protein kinase, RLK)基因TaPK3A, 并对其表达特性及抗纹枯病功能进行了分析和验证。TaPK3A基因的开放阅读框长度为1983 bp, 编码660个氨基酸组成的类受体蛋白激酶。TaPK3A在抗纹枯病小麦品系CI12633中的表达受禾谷丝核菌的诱导而显著上调; TaPK3A在根、茎、叶、穗中都有表达, 以叶中的表达量最高; TaPK3A的表达受植物激素水杨酸诱导最为显著。利用大麦条形花叶病毒(barley stripe mosaic virus, BSMV)诱导的基因沉默(virus-induced gene silencing, VIGS)技术, 降低抗纹枯病小麦CI12633中TaPK3A的转录水平, 再接种禾谷丝核菌WK207进行纹枯病抗性鉴定。结果显示, 与对照植株相比, TaPK3A转录量下降的CI12633植株对纹枯病的抗性显著降低, 说明TaPK3A是小麦防御纹枯病反应所需的。

关键词: 类受体蛋白激酶TaPK3A, 小麦纹枯病, 抗病反应, 病毒诱导的基因沉默

Abstract:

Wheat sharp eyespot has become an important limiting factor of wheat production in China. The precondition for wheat sharp eyespot resistant breeding is to study the molecular basis of wheat defense response and to identify effective resistance genes. In this study, a wheat receptor-like kinase (RLK) gene, named TaPK3A, was cloned from sharp eyespot-resistant wheat line CI12633, and the expression and function of the TaPK3A gene were analyzed. TaPK3A contains an open reading frame with 1983 bp length. It encodes a protein kinase that is consisted of 660 amino acids. RT-qPCR analysis showed that the expression of TaPK3A in sharp eyespot-resistant wheat line CI12633 was significantly induced by the pathogen of sharp eyespot (Rhizoctonia cerealis). The TaPK3A gene was expressed in all the tissues, with the highest expression level in the leaves. The expression of TaPK3A was significantly up-regulated by salicylic acid. By means of barley stripe mosaic virus (BSMV) based virus-induced gene-silencing (VIGS), TaPK3A was silenced in CI12633 plants. After R. cerealis (WK207) inoculation, TaPK3A-silenced CI12633 plants displayed a significant decrease in resistance to R. cerealis infection compared with BSMV: GFP-infected CI12633 plants (control). These results suggested that TaPK3A is required for wheat defense response to sharp eyespot.

Key words: receptor-like kinase TaPK3A, wheat sharp eyespot, resistance response, virus-induced gene silencing

图1

RIL中TaPK3A的表达分析 A: 抗感材料RNA-Seq 数据中TaPK3A的表达量; B: RT-qPCR验证TaPK3A的表达量; R代表RIL中抗纹枯病小麦株系, S代表RIL中感纹枯病株系; R0/S0表示在0 h时R中和S中TaPK3A的表达量的比值; R4/S4表示在4 d时R中和S中TaPK3A的表达量的比值; R10/S10表示在10 d时R中和S中TaPK3A的表达量的比值。"

图2

TaPK3A蛋白的系统进化树分析 用MEGA7对TaPK3A进化树分析, 五角星标注的是TaPK3A蛋白, 竖线内为同一亚家族蛋白。AtCERK1 (NP_566689.2)、AtLecRK-VI.2 (Q9M021.1)、AtLPK1 (NP_567233.1)、AtPSKR1 (OAP07882.1)、AtWAK1 (OAP13193.1)、AtWAKL10 (NP_178086.1)来自拟南芥; OnN25-1 (AHW98543.1)来自野生稻; NbLRK1 (BAG68210.1)来自本氏烟; OsWAK1 (BAG89532.1)、OsCERK1 (A0A0P0XII1.1)、OsXa26 (AAR08150.1)来自水稻; Sbds1 (BAM45642.1)来自高粱; SiBti9 (ADL16642.1)来自谷子; TaLRK10 (AAC49629.1)来自小麦; ZmPK3 (CAA09029.1)来自玉米。"

图3

小麦品系CI12633中TaPK3A基因的表达分析 A: TaPK3A基因在抗纹枯病小麦CI12633中的表达分析, 横坐标表示禾谷丝核菌侵染小麦CI12633后0 h、2 d、4 d、7 d、10 d、14 d和21 d时的样品。B: CI12633小麦不同组织中TaPK3A受禾谷丝核菌的诱导表达分析。C: TaPK3A基因响应 SA、JA、ABA和 ET的表达分析, 横坐标表示不同植物激素处理小麦CI12633后0 h、0.5 h、1 h、6 h和24 h时的样品。t检验分析TaPK3A表达量的差异显著性, *P < 0.05; **P < 0.01。"

图4

BSMV CP、TaPK3A基因的检测及小麦花叶和纹枯病表型 A: 半定量PCR检测CP的转录表达, 扩增26个循环; B: 对接种BSMV: GFP和BSMV: TaPK3A 10 d后的植株RT-qPCR检测TaPK3A的表达, **P < 0.01 (t检验); C: 接种BSMV后10 d, 小麦叶片呈现出BSMV病毒斑; D: 接种禾谷丝核菌后30 d小麦茎秆上纹枯病斑, IT: 病级"

表1

TaPK3A沉默及对照小麦的纹枯病鉴定"

基因型
Genotype		 病级
Infection type		 病情指数
Disease index
BSMV:TaPK3A 2.31** 46.2**
BSMV:GFP 1.27 25.4
[1] 王敏霞, 祝秀亮, 罗美英, 张增艳 . 小麦防御素基因TaPDF35的克隆与功能分析. 植物遗传资源学报, 2017,18:925-932.
Wang M X, Zhu X L, Luo M Y, Zhang Z Y . Cloning and defensive functional analysis of a wheat defensin gene TaPDF35. J Plant Genet Resour, 2017,18:925-932 (in Chinese with English abstract).
[2] 史建荣, 王裕中, 陈怀谷, 沈素文 . 小麦纹枯病品种抗性鉴定技术及抗病资源的筛选与分析. 植物保护学报, 2000,27:107-112.
Shi J R, Wang Y Z, Chen H G, Shen S W . Screening techniques and evaluation of wheat resistance to sharp eyespot caused by Rhizoctonia cerealis. Acta Phytophyl Sin, 2000,27:107-112 (in Chinese with English abstract).
[3] 梁邦平, 郝冬冬, 刁慧珊, 李家创, 袁凤平, 李毛, 武军, 赵继新, 陈新宏 . 小麦-黑麦1BL/1RS易位系7-1抗纹枯病的分子细胞学鉴定. 农业生物技术学报, 2018,26:711-718.
Liang B P, Hao D D, Diao H S, Li J C, Yuan F P, Li M, Wu J, Zhao J X, Chen X H . Molecular cytogenetic identification of wheat-rye (Triticum aestivum-Secale cereale) 1BL/1R translocation line 7-1 with sharp eyespot resistance. J Agric Biotechnol, 2018,26:711-718 (in Chinese with English abstract).
[4] Jones J D, Dangl J L . The plant immune system. Nature, 2006,444:323-329.
doi: 10.1038/nature05286
[5] Morris E R, Walker J C . Receptor-like protein kinases: the keys to response. Curr Opin Plant Biol, 2003,6:339-342.
doi: 10.1016/S1369-5266(03)00055-4
[6] Shiu S H, Bleecker A B . Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci USA, 2001,98:10763-10768.
[7] Walker J C . Structure and function of the receptor-like protein kinases of higher plants. Plant Mol Biol, 1994,26:1599-1609.
doi: 10.1007/BF00016492
[8] Tor M, Lotze M T, Holton N . Receptor-mediated signalling in plants: molecular patterns and programmes. J Exp Bot, 2009,60:3645-3654.
doi: 10.1093/jxb/erp233
[9] Ma C L, Guo J, Kang Y, Doman K, Bryan A C, Tax F E, Yamaguchi Y, Qi Z . AtPEPTIDE RECEPTOR2 mediates the AtPEPTIDE1-induced cytosolic Ca2+ rise, which is required for the suppression of Glutamine Dumper gene expression in Arabidopsis roots. J Integr Plant Biol, 2014,56:684-694.
[10] Shiu S H, Karlowski W M, Pan R S, Tzeng Y H, Mayer K F X, Li W H . Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell, 2004,16:1220-1234.
[11] Verica J A, He Z H . The cell wall-associated kinase (WAK) and WAK-like kinase gene family. Plant Physiol, 2002,129:455-459.
doi: 10.1104/pp.011028
[12] Chang C, Kwok S F, Bleecker A B, Meyerowitz E M . Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science, 1993,262:539-544.
[13] Becraft P W, Stinard P S, McCarty D R . CRINKLY4: A TNFR-like receptor kinase involved in maize epidermal differentiation. Science, 1996,273:1406-1409.
doi: 10.1126/science.273.5280.1406
[14] Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K . The Sreceptor kinase determines self-incompatibility in Brassica stigma. Nature, 2000,403:913-916.
[15] Endre G, Kereszt A, Kevei Z, Mihacea S, Kalo P, Kiss G B . A receptor kinase gene regulating symbiotic nodule development. Nature, 2002,417:962-926.
[16] Nishimura R, Hayashi M, Wu G J, Kouchi H, Imaizumi-Anraku H, Murakami Y, Kawasaki S, Akao S, Ohmori M, Nagasawa M, Harada K, Kawaguchi M . HAR1 mediates systemic regulation of symbiotic organ development. Nature, 2002,420:426-429.
[17] Loh Y T, Martin G B . The Pto bacterial resistance gene and the Fen insecticide sensitivity gene encode functional protein kinases with serine/threonine specificity. Plant Physiol, 1995,108:1735-1739.
[18] Song W Y, Wang G L, Chen L L, Kim H S, Pi L Y, Holsten T, Gardner J, Wang B, Zhai W X, Zhu L H, Fauquet C, Ronald P . A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science, 1995,270:1804-1806.
[19] Sun X, Cao Y, Yang Z, Xu C, Li X, Wang S, Zhang Q . Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein. Plant J, 2004,37:517-527.
[20] Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G . A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci USA, 2010,107:9452-9457.
doi: 10.1073/pnas.1000675107
[21] Kohorn B D, Johansen S, Shishido A, Todorova T, Martinez R, Defeo E, Obregon P . Pectin activation of MAP kinase and gene expression is WAK2 dependent. Plant J, 2009,60:974-982.
doi: 10.1111/tpj.2009.60.issue-6
[22] Zuo W, Chao Q, Zhang N, Ye J, Tan G, Li B, Xing Y, Zhang B, Liu H, Fengler K A, Zhao J, Zhao X, Chen Y, Lai J, Yan J, Xu M . A maize wall-associated kinase confers quantitative resistance to head smut. Nat Genet, 2015,47:151-157.
[23] Singh P, Kuo Y C, Mishra S, Tsai C H, Chien C C, Chen C W, Desclos-Theveniau M, Chu P W, Schulze B, Chinchilla D, Boller T, Zimmerli L . The lectin receptor kinase-VI.2 is required for priming and positively regulates Arabidopsis pattern-triggered immunity. Plant Cell, 2012,24:1256-1270.
[24] Chen X, Shang J, Chen D, Lei C, Zou Y, Zhai W, Liu G, Xu J, Ling Z, Cao G, Ma B, Wang Y, Zhao X, Li S, Zhu L . A B-lectin receptor kinase gene conferring rice blast resistance. Plant J, 2006,46:794-804.
doi: 10.1111/tpj.2006.46.issue-5
[25] Liu Y, Wu H, Chen H, Liu Y, He J, Kang H, Sun Z, Pan G, Wang Q, Hu J, Zhou F, Zhou K, Zheng X, Ren Y, Chen L, Wang Y, Zhao Z, Lin Q, Wu F, Zhang X, Guo X, Cheng X, Jiang L, Wu C, Wang H, Wan J . A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice. Nat Biotechnol, 2015,33:301-305.
doi: 10.1038/nbt.3069
[26] Feuillet C, Schachermayr G, Keller B . Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat. Plant J, 2010,11:45-52.
[27] Zhou H B, Li S F, Deng Z Y, Wang X P, Chen T, Zhang J S, Chen S Y, Ling H Q, Zhang A M, Wang D W, Zhang X Q . Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection. Plant J, 2007,52:420-434.
doi: 10.1111/j.1365-313X.2007.03246.x
[28] Livak K J, Schmittgen T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 2001,25:402-408
doi: 10.1006/meth.2001.1262
[29] Holzberg S, Brosio P, Gross C, Pogue G P . Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J, 2002,30:315-327.
[30] Zhu X L, Yang K, Wei X N, Zhang Q F, Rong W, Du L P, Ye X G, Qi L, Zhang Z Y . The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis. J Exp Bot, 2015,66:6591-6603.
[31] 周淼平, 杨学明, 姚金保, 任丽娟, 张增艳, 马鸿翔 . 转Gastrodianin基因提高小麦赤霉病和纹枯病的抗性. 作物学报, 2012,38:1617-1624.
Zhou M P, Yang X M, Yao J B, Ren L J, Zhang Z Y, Ma H X . Enhancement of resistance to Fusarium head blight and sharp eyespot in Gastrodianin transgenic wheat. Acta Agron Sin, 2012,38:1617-1624 (in Chinese with English abstract).
[32] Sanabria N, Goring D, Nurnberger T, Dubery I . Self/nonself perception and recognition mechanisms in plants: a comparison of self-incompatibility and innate immunity. New Phytologist, 2008,178:503-513.
doi: 10.1111/nph.2008.178.issue-3
[33] Brueggeman R, Rostoks N, Kudrna D, Kilian A, Han F, Chen J, Druka A, Steffenson B, Kleinhofs A . The barley stem rust- resistance gene Rpg1 is a novel disease-resistance gene with homology to receptor kinases. Proc Natl Acad Sci USA, 2002,99:9328-9333.
[34] Ahmed S M, Liu P, Xue Q, Ji C, Qi T, Guo J, Guo J, Kang Z . TaDIR1-2, a wheat ortholog of lipid transfer protein AtDIR1 contributes to negative regulation of wheat resistance against Puccinia striiformis f. sp. tritici. Front Plant Sci, 2017,8:521.
[35] Kage U, Karre S, Kushalappa A C, McCartney C . Identification and characterization of a fusarium head blight resistance gene TaACT in wheat QTL-2DL. Plant Biotechnol J, 2017,15:447-457.
[36] Liu J, Zhang T, Jia J, Sun J . The wheat mediator subunit TaMED25 interacts with the transcription factor TaEIL1 to negatively regulate disease resistance against powdery mildew. Plant Physiol, 2016,170:1799-1816.
doi: 10.1104/pp.15.01784
[37] Zou B, Ding Y, Liu H, Hua J . Silencing of copine genes confers common wheat enhanced resistance to powdery mildew. Mol Plant Pathol, 2017,19:1343-1352.
[38] Scofield S R, Huang L, Brandt A S, Gill B S . Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol, 2005,138:2165-2173.
[39] Zhu X, Lu C, Du L, Ye X, Liu X, Coules A, Zhang Z . The wheat NB-LRR gene TaRCR1 is required for host defence response to the necrotrophic fungal pathogen Rhizoctonia cerealis. Plant Biotechnol J, 2017,15:674-687.
[40] Dong X . SA, JA, ethylene, and disease resistance in plants. Curr Opin Plant Biol, 1998,1:316-323.
doi: 10.1016/1369-5266(88)80053-0
[41] Glazebrook J . Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol, 2005,43:205-227
doi: 10.1146/annurev.phyto.43.040204.135923
[42] Chen T, Xiao J, Xu J, Wan W, Qin B, Cao A, Chen W, Xing L, Du C, Gao X, Zhang S, Zhang R, Shen W, Wang H, Wang X . Two members of TaRLK family confer powdery mildew resistance in common wheat. BMC Plant Biol, 2016,16:27.
[1] 申芳嫡,洪彦涛,杜丽璞,徐惠君,马翎健,张增艳. 转细胞凋亡抑制基因OpIAP和p35增强小麦对纹枯病的抗性[J]. 作物学报, 2015, 41(10): 1490-1499.
[2] 王金凤，杜丽璞，李钊，黄素萍，叶兴国，冯斗，张增艳. 抗纹枯病、根腐病的转SN1基因小麦的获得与鉴定[J]. 作物学报, 2012, 38(05): 773-779.
[3] 赵丹, 赵继荣, 黄茜, 李宁, 黄占景, 张增艳. 利用BSMV-VIGS技术快速分析小麦TNBL1基因的抗黄矮病功能[J]. 作物学报, 2011, 37(11): 2106-2110.
Viewed
Full text


Abstract

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