海島棉GbWRKY40基因的克隆及特征分析

倪志勇，加得拉·吐留汗，邱迎風(fēng)，曲延英，陳全家

（新疆農(nóng)業(yè)大學(xué)農(nóng)學(xué)院／新疆農(nóng)業(yè)大學(xué)農(nóng)業(yè)生物技術(shù)重點(diǎn)實(shí)驗(yàn)室，烏魯木齊830052）

海島棉GbWRKY40基因的克隆及特征分析

倪志勇，加得拉·吐留汗，邱迎風(fēng)，曲延英，陳全家*

（新疆農(nóng)業(yè)大學(xué)農(nóng)學(xué)院／新疆農(nóng)業(yè)大學(xué)農(nóng)業(yè)生物技術(shù)重點(diǎn)實(shí)驗(yàn)室，烏魯木齊830052）

【目的】WRKY轉(zhuǎn)錄因子調(diào)控多種生物學(xué)進(jìn)程，包括植物生長發(fā)育和應(yīng)答多種環(huán)境脅迫。本研究旨在分析WRKY轉(zhuǎn)錄因子在海島棉纖維發(fā)育中的功能?！痉椒ā繌暮u棉中克隆了1個(gè)WRKY轉(zhuǎn)錄因子基因Gb-WRKY40，進(jìn)行同源性分析、多序列比對(duì)，利用熒光定量聚合酶鏈?zhǔn)椒磻?yīng)分析其表達(dá)模式，通過構(gòu)建酵母表達(dá)載體并轉(zhuǎn)化酵母菌株AH109研究其轉(zhuǎn)錄激活活性?！窘Y(jié)果】該基因cDNA全長1713 bp，5'非編碼區(qū)長261 bp，3'非編碼區(qū)長510 bp；開放閱讀框長942 bp，編碼313個(gè)氨基酸，預(yù)測相對(duì)分子質(zhì)量約為34.138×103，等電點(diǎn)為8.46，包含5個(gè)外顯子和4個(gè)內(nèi)含子；其編碼蛋白含有1個(gè)WRKY保守區(qū) （WRKYGQK）和1個(gè)鋅指基序（C-X5-C-X23-H-X1-H），屬于WRKY家族第Ⅱ類a組，包含3個(gè)核定位信號(hào)區(qū)，與陸地棉GhWRKY40同源性最高。GbWRKY40在根和開花后25 d纖維中表達(dá)量高，而且不具有轉(zhuǎn)錄激活活性。【結(jié)論】GbWRKY40可能參與調(diào)控棉纖維次生壁發(fā)育。

海島棉；GbWRKY40；表達(dá)模式；基因克??；轉(zhuǎn)錄激活

植物WRKY轉(zhuǎn)錄因子形成1個(gè)大的轉(zhuǎn)錄因子家族，在植物應(yīng)答逆境脅迫、表皮毛發(fā)育和次生代謝中具有重要的功能[1]。WRKY蛋白包含1個(gè)或2個(gè)DNA結(jié)合結(jié)構(gòu)域，每個(gè)結(jié)構(gòu)域由60個(gè)氨基酸組成，其N端存在1個(gè)高度保守的WRKYGQK序列，C端具有1個(gè)鋅指類似基序。根據(jù)含有的WRKY結(jié)構(gòu)域數(shù)量和鋅指類似基序的特點(diǎn)，WRKY轉(zhuǎn)錄因子可分為3類：多數(shù)蛋白具有1個(gè)WRKY結(jié)構(gòu)域，屬于第Ⅱ類，具有2個(gè)WRKY結(jié)構(gòu)域的蛋白屬于第Ⅰ類，通常這2類WRKY蛋白具有相同的鋅指類似基序C-X4-5-C-X22-23-H-X1-H；第Ⅲ類WRKY蛋白成員具有1個(gè)WRKY結(jié)構(gòu)域，其鋅指類似基序?yàn)镃-X7-C-X23-H-X-C[1]。

隨著不同棉種基因組測序的完成[2-7]，不同棉種WRKY轉(zhuǎn)錄因子的鑒定成為研究熱點(diǎn)，雷蒙德氏棉 （Gossypium raimondii）、亞洲棉 （G.arboreum）和陸地棉（G.hirsutum）中分別發(fā)現(xiàn)116個(gè)、109個(gè)和 102個(gè)WRKY基因[8-10]。陸地棉WRKY轉(zhuǎn)錄因子的研究較多，許多陸地棉WRKY基因參與調(diào)控植物對(duì)生物和非生物脅迫的應(yīng)答。一些WRKY轉(zhuǎn)錄因子是正調(diào)控子，例如在煙草中過表達(dá)GhWRKY41基因，能夠提高轉(zhuǎn)基因植株對(duì)干旱和鹽脅迫的忍耐能力[11]。相比野生型植株，過表達(dá)GhWRKY34的擬南芥耐鹽能力增強(qiáng)[12]。過表達(dá)GhWRKY39-1的轉(zhuǎn)基因煙草對(duì)病原菌侵染和鹽脅迫的忍耐力增強(qiáng)[13]。相反，一些WRKY轉(zhuǎn)錄因子起負(fù)調(diào)控子作用。例如，相比野生型煙草，過表達(dá)GhWRKY27a的煙草對(duì)干旱和立枯絲核菌的抵抗能力降低[14]。GhWRKY68[15]和GhWRKY17[16]降低轉(zhuǎn)基因煙草對(duì)鹽和干旱的忍耐能力。相比野生型煙草，過表達(dá)GhWRKY15的轉(zhuǎn)基因煙草對(duì)病毒和真菌侵染敏感[17]。此外，有些WRKY轉(zhuǎn)錄因子在不同的脅迫應(yīng)答中功能不同：例如在煙草中過表達(dá)GhWRKY25會(huì)降低植物對(duì)干旱脅迫的忍耐能力，但增強(qiáng)了其耐鹽能力，轉(zhuǎn)基因植株對(duì)葡萄孢菌敏感[18]；過表達(dá)Gh-WRKY40增強(qiáng)轉(zhuǎn)基因煙草的傷害耐受力和對(duì)青枯病的敏感性[19]。陸地棉WRKY轉(zhuǎn)錄因子也參與調(diào)控植物衰老過程，例如GhWRKY11延緩轉(zhuǎn)基因擬南芥衰老[20]。相比陸地棉，其他棉種WRKY基因報(bào)道較少。最近，F(xiàn)an等利用轉(zhuǎn)錄組分析方法從耐鹽的棉花野生種旱地棉（G.aridum）中鑒定了109個(gè)WRKY基因，過表達(dá)GarWRKY17和GarWRKY104基因的轉(zhuǎn)基因擬南芥耐鹽性增強(qiáng)[21]。亞洲棉GaWRKY1參與調(diào)控倍半萜烯代謝[22]。海島棉（G.barbadenceL.）GbWRKY1是磷缺乏應(yīng)答的正調(diào)控子[23]。

關(guān)于海島棉WRKY轉(zhuǎn)錄因子研究的報(bào)道不多，WRKY轉(zhuǎn)錄因子在海島棉纖維發(fā)育中的作用仍不清楚。本研究從海島棉中克隆到1個(gè)棉花WRKY類轉(zhuǎn)錄因子基因GbWRKY40，分析了該基因的序列、轉(zhuǎn)錄激活活性及表達(dá)模式。

1 材料與方法

1.1 植物材料

以海島棉新海21（Xinhai 21）為供試品種，取盛花期的苞葉、花、根、莖、葉及不同發(fā)育階段的棉纖維，液氮速凍，－80℃保存?zhèn)溆谩?/p>

1.2 RNA的提取和cDNA第1鏈的合成

按RNA plant plus reagent試劑盒（Tiangen）說明書操作步驟，提取苞葉、花、根、莖、葉組織總RNA。按多糖多酚植物總RNA提取試劑盒（Tiangen）說明書操作步驟，提取不同發(fā)育階段的棉纖維總RNA。使用DNaseⅠ（Thermo）除去總RNA中的基因組DNA。利用First Strand cDNA Synthesis試劑盒（Thermo）將各組織總RNA反轉(zhuǎn)錄合成cDNA第1鏈。

1.3GbWRKY40基因的克隆及序列分析

根據(jù)本課題組前期海島棉胚珠轉(zhuǎn)錄組和纖維不同發(fā)育表達(dá)譜數(shù)據(jù)，共獲得131個(gè)WRKY unigene序列，選取1個(gè)unigene序列CL2113.Contig3_All設(shè)計(jì) 1對(duì)引物，GbW40-F：5'-ATGGAATCGACTTGGGTGGATACGA-3'和GbW40-R: 5'-TTACCACTTGTGATCTAGAACTTTTCCAGAA-3'。以合成的棉花葉片cDNA第1鏈為模板，用TransStart Taq DNA酶擴(kuò)增 （TransGen）獲得GbWRKY40的cDNA序列?；厥占兓疨CR（Polymerase chain reaction） 產(chǎn) 物 ， 連 接 到pMD19-T載體（TaKaRa），采用菌液PCR方法鑒定獲得陽性克隆，送上海美吉公司測序。

利用DNAMAN7進(jìn)行同源性分析，利用Clustalx1.83軟件進(jìn)行多序列比對(duì)，利用MEGA4.1軟件構(gòu)建系統(tǒng)發(fā)生樹。

1.4 實(shí)時(shí)定量PCR

實(shí)時(shí)定量PCR（Real-time quantitative PCR，RT-PCR）使用Maxima SYBR Green/ROX qPCR Master Mix(2×)試劑盒（Fermentas），GbWRKY40引物序列為，GbW40-qF：5'-AGCCTGATTGTGAGAGATGGAT-3'和GbW40-qR：5'-TTTCTTGACGGGACAACTTGG-3'。GbUBQ7為內(nèi)參基因，引物序列為，UBQ7-F：5'-GACCTACACCAAGCCCAAGAAG-3'和UBQ7-R：5'-TGAGCCCACACTTACC ACAATAGT-3'。按照試劑盒說明書推薦體系和反應(yīng)程序操作。使用ABI 7500 Fast實(shí)時(shí)熒光定量PCR系統(tǒng)進(jìn)行實(shí)時(shí)定量PCR。基因的相對(duì)表達(dá)量采用方法分析。試驗(yàn)進(jìn)行3次生物學(xué)重復(fù)，每組生物學(xué)重復(fù)做3次技術(shù)重復(fù)。數(shù)據(jù)采用SPSS 17.0（SPSS Inc.,USA）最小顯著差數(shù)法（Least significant difference，LSD）進(jìn)行差異顯著性分析。

1.5 酵母轉(zhuǎn)化

使用帶有EcoRⅠ和BamHⅠ酶切位點(diǎn)的引物擴(kuò)增GbWRKY40的開放閱讀框序列，引物序列為：w40-F：5'-TTTGAATTCATGGAATCGACTTGGGTGGATACGA-3'，w40-R：5'-TTTGGATCCTTACCACTTGTGATCTAGAACTTTTCCAGA-3'，下劃線表示酶切位點(diǎn)。凝膠回收帶有酶切位點(diǎn)GbWRKY40的開放閱讀框序列，雙酶切后亞克隆至pGBKT7載體中，通過測序驗(yàn)證載體構(gòu)建的正確性。按照Frozen-EZ Yeast TransformationⅡ試劑盒（ZYMO RESEARCH，Orange,CA）說明書操作步驟，將GbWRKY40-pGBKT7、pGBKT7、陽性對(duì)照GmNAC115-pGBKT7質(zhì)粒[24]轉(zhuǎn)化酵母菌株AH109，涂布于單缺SD/-Trp營養(yǎng)缺陷型培養(yǎng)基（SD/-Trp）和三缺SD/-Trp/-His/-Ade營養(yǎng)缺陷型培養(yǎng)基 （SD/-Trp/-His/-Ade），30℃培養(yǎng)2～3 d后，觀察菌落的生長情況。

2 結(jié)果與分析

2.1GbWRKY40基因全長cDNA克隆及序列分析

根據(jù)本課題組海島棉胚珠轉(zhuǎn)錄組和纖維不同發(fā)育表達(dá)譜數(shù)據(jù)得到的棉花WRKY相關(guān)unigene序列CL2113.Contig3_All，用RT-PCR方法，獲得全長cDNA序列，將其命名為GbWRKY40。GbWRKY40全長cDNA為1 713 bp，5'端非編碼區(qū)261 bp，3'非編碼區(qū)為510 bp；編碼區(qū)開放閱讀框長942 bp，編碼313個(gè)氨基酸，預(yù)測相對(duì)分子質(zhì)量約為34.138×103，等電點(diǎn)為8.46。氨基酸序列分析表明該蛋白含有1個(gè)WRKY保守區(qū)，位于第 156～212個(gè) 氨基酸 ，1個(gè)鋅 指 基 序C-X5-C-X23-H-X1-H，3個(gè)核定位信號(hào)區(qū)，暗示該轉(zhuǎn)錄因子可能定位于細(xì)胞核（圖1）。系統(tǒng)發(fā)生樹分析表明GbWRKY40屬于WRKY家族第Ⅱ類a組（圖 2）。

同源性分析表明，GbWRKY40與TcWRKY40、CcWRKY40、PtWRKY40和PeWRKY的氨基酸序列一致性分別為72.87%，55.86%，56.48%和55.69%。在棉屬中，GbWRKY40與同為四倍體的陸地棉GhWRKY40的氨基酸序列一致性最高（99.04%），與亞洲棉GaWRKY40和草棉 Gr-WRKY40的氨基酸序列一致性分別為81.96%和95.21%。

海島棉基因組數(shù)據(jù)庫比對(duì)發(fā)現(xiàn)，GbWRKY40基因定位于 Gbscaffold17271.6.0 scaffold17271: 72937～74804(+strand)，GbWRKY40基因全長1868 bp，包含5個(gè)外顯子和4個(gè)內(nèi)含子。

2.2 GbWRKY40的表達(dá)模式分析

利用熒光定量PCR方法分析GbWRKY40在海島棉不同組織中的表達(dá)情況，結(jié)果表明在所檢測的組織中GbWRKY40在根中表達(dá)量最高，在莖中最低，其他組織中的表達(dá)量依次為葉、花和苞葉（圖3），暗示該基因在這些組織中的表達(dá)水平可能與這些組織行使的生物學(xué)功能有關(guān)。GbWRKY40在開花后5 d的纖維中表達(dá)量最低，在開花后25 d的纖維中表達(dá)量最高（圖4），推測該基因可能在棉纖維次生壁合成過程中發(fā)揮作用。

圖1 GbWRKY40與其他已知WRKY蛋白的氨基酸序列比對(duì)Fig.1 Alignment of the amino acid sequences of GbWRKY40 and other known WRKY homologs proteins

2.3 GbWRKY40轉(zhuǎn)錄激活活性分析

用GbWRKY40-pGBKT7、pGBKT7和Gm-NAC115-pGBKT7轉(zhuǎn)化酵母菌株AH109，并將其涂布于單缺SD/-Trp營養(yǎng)型缺陷培養(yǎng)基后觀察發(fā)現(xiàn)，轉(zhuǎn)化GbWRKY40-pGBKT7、pGBKT7和Gm-NAC115-pGBKT7的酵母菌落都能正常生長 （圖5），說明上述3個(gè)質(zhì)粒均已轉(zhuǎn)化到酵母中。而在三缺SD/-Trp/-His/-Ade營養(yǎng)型缺陷培養(yǎng)基上，轉(zhuǎn)化GbWRKY40-pGBKT7的酵母菌落和陰性對(duì)照pGBKT7的酵母菌落均不能正常生長，而陽性對(duì)照GmNAC115-pGBKT7的酵母菌落能正常生長（圖5），說明GbWRKY40不具有轉(zhuǎn)錄激活活性，推測GbWRKY40可能是1個(gè)轉(zhuǎn)錄抑制子。

3 討論

本研究從海島棉中克隆了1個(gè)WRKY類轉(zhuǎn)錄因子基因GbWRKY40，氨基酸序列和進(jìn)化樹分析發(fā)現(xiàn)GbWRKY40含有1個(gè)WRKY保守區(qū)和 1個(gè)鋅指基序 C-X5-C-X23-H-X1-H，屬于WRKY轉(zhuǎn)錄因子家族第Ⅱ類a組。不同物種的同一亞族成員間可能具有類似的功能，GbWRKY40與擬南芥AtWRKY18/40/60同屬WRKY家族第Ⅱ類a組，之前研究發(fā)現(xiàn)AtWRKY18/40/60形成1個(gè)高度互作的調(diào)控網(wǎng)絡(luò)，通過轉(zhuǎn)錄激活或抑制調(diào)控植物防御和非生物脅迫應(yīng)答基因的表達(dá)，在植物逆境脅迫應(yīng)答中具有重要的功能[25-26]。Gb-WRKY40轉(zhuǎn)錄因子是否參與調(diào)控植物逆境脅迫響應(yīng)需要進(jìn)一步研究。

圖2 GbWRKY40蛋白系統(tǒng)發(fā)生樹分析Fig.2 Phylogenetic analysis of the GbWRKY40 protein

圖3GbWRKY40基因在不同組織中的表達(dá)模式Fig.3 Expression profile ofGbWRKY40 gene in different tissues

圖4GbWRKY40在不同纖維發(fā)育階段的表達(dá)模式Fig.4 Expression profile ofGbWRKY40 gene at different fiber developmental stages

圖5 GbWRKY40轉(zhuǎn)錄因子的轉(zhuǎn)錄激活試驗(yàn)Fig.5 Transcriptional activation ability assay of the GbWRKY40 transcription factor

前人研究表明棉花不同的WRKY轉(zhuǎn)錄因子可以作為轉(zhuǎn)錄激活子或抑制子調(diào)控下游基因的表達(dá)，對(duì)新克隆的WRKY轉(zhuǎn)錄因子進(jìn)行轉(zhuǎn)錄激活活性分析有助于了解WRKY轉(zhuǎn)錄因子的調(diào)控功能。酵母系統(tǒng)是常用的研究轉(zhuǎn)錄因子轉(zhuǎn)錄激活活性的體內(nèi)研究方法。例如，通過轉(zhuǎn)化酵母細(xì)胞發(fā)現(xiàn)GhWRKY31/33[27]、GhWRKY40[19]、GhWRKY41[11]具有轉(zhuǎn)錄激活活性，而GhWRKY12/16/32/34[27]和海島棉GbWRKY32[28]不具有轉(zhuǎn)錄激活效應(yīng)。本研究通過酵母系統(tǒng)發(fā)現(xiàn)GbWRKY40不具有轉(zhuǎn)錄激活活性，與GbWRKY40屬于同一亞組的擬南芥AtWRKY40是轉(zhuǎn)錄抑制子，而AtWRKY18/60是弱轉(zhuǎn)錄激活子[26]；因此，推測GbWRKY40可能是1個(gè)轉(zhuǎn)錄抑制子。下一步將通過擬南芥原生質(zhì)體系統(tǒng)驗(yàn)證GbWRKY40對(duì)下游基因的抑制作用。

目前，已報(bào)道的棉花WRKY轉(zhuǎn)錄因子主要參與逆境脅迫應(yīng)答和衰老進(jìn)程[14-20,29]，參與棉纖維發(fā)育的WRKY轉(zhuǎn)錄因子報(bào)道不多。Wan等研究表明棉纖維的發(fā)育可能與擬南芥的表皮毛發(fā)育具有類似的分子機(jī)制[30]，擬南芥TTG2編碼1個(gè)WRKY轉(zhuǎn)錄因子，激活下游表皮毛起始基因TTG1和GL1[31]，這些研究表明WRKY轉(zhuǎn)錄因子可能在棉纖維發(fā)育過程中具有一定的功能。周立研究發(fā)現(xiàn)GhWRKY12和GhWRKY16在開花后3～10 d的棉纖維中優(yōu)勢表達(dá)，推測這些基因可能在棉纖維發(fā)育的起始和伸長期發(fā)揮一定作用[27]。本研究發(fā)現(xiàn)GbWRKY40在根和開花后25 d的纖維中優(yōu)勢表達(dá)，推測該基因可能在棉纖維次生壁發(fā)育中具有一定的功能，下一步將通過轉(zhuǎn)化海島棉來研究基因的生物學(xué)功能。

4 結(jié)論

從海島棉中克隆了1個(gè)WRKY轉(zhuǎn)錄因子基因GbWRKY40，其不具有轉(zhuǎn)錄激活活性，在根和開花后25 d的纖維中表達(dá)量高。推測該基因可能參與調(diào)控棉纖維次生壁發(fā)育。

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Cloning and Characterization of theGbWRKY40 Transcription Factor Gene fromGossypium barbadenseL.

Ni Zhiyong,Jiadela Tuliuhan,Qiu Yingfeng,Qu Yanying,Chen Quanjia*
(College of Agronomy,Xinjiang Agricultural University/Key Laboratory of Agricultural Biological Technology,Xinjiang Agricultural University,Urumqi830052,China)

[Objective]The WRKY transcription factors modulate various biological processes,including plant growth,development,and responses to various environmental stresses.This study aimed to clarify the role of WRKY transcription factors related to cotton fiber development.[Method]TheGbWRKY40 gene,which encodes a WRKY transcription factor,was isolated from cotton(Gossypium barbadenseL.).The obtained sequence was analyzed for homology with genes from other cotton species using the DNAMAN7 program.The sequences of the homologous genes were aligned with Clustalx1.83.Additionally,theGb-WRKY40 expression pattern was analyzed by a quantitative real-time polymerase chain reaction.Furthermore,a yeast expression vector was constructed and inserted into yeast strain AH109 cells to investigate the transcriptional activity ofGbWRKY40.[Result]Full-lengthGbWRKY40 cDNA contains 1713 nucleotides,including a 942 bp open reading frame,a 261 bp 5'-untranslated region,and a 510 bp 3'-untranslated region.The open reading frame was predicted to encode a 313 amino acid protein with a relative molecular mass of 34.138×103and an isoelectric point of 8.46.The genomicGbWRKY40 sequence was observed to comprise five exons and four introns.The predicted GbWRKY40 protein includes a WRKY domain consisting of approximately 60 amino acids,including the conserved WRKYGQK sequence and a zinc-finger motif(C-X5-C-X23-H-X1-H).These characteristics indicated that GbWRKY40 belongs to group IIa of the WRKY family.Moreover,GbWRKY40 contains three putative nuclear localization signals.GbWRKY40 is highly homologous toGhWRKY40.Expression analyses involving a quantitative real-time polymerase chain reaction revealed thatGbWRKY40 is highly expressed in cotton roots and fibers at 25 days post anthesis.Transcriptional activation assay results suggested that the function of the GbWRKY40 transcription factor is unrelated to the activation of transcription.[Conclusion]These results imply thatGbWRKY40 may be involved in regulating secondary cell walldevelopment in cotton fibers.

cotton;GbWRKY40;expression profile;gene clone;transcriptional activation

S562.035

A

1002-7807（2017）04-0393-08

10.11963/1002-7807.nzycqj.20170601

2016-10-13

倪志勇（1981―），男，博士，nizhiyong@126.com。*通信作者：chqjia@126.com

新疆維吾爾自治區(qū)青年科技創(chuàng)新人才培養(yǎng)工程優(yōu)秀青年科技人才（qn2015yx012）