中國農業科學 ?? 2019, Vol. 52 ?? Issue (11): 1858-1869.doi: 10.3864/j.issn.0578-1752.2019.11.002

? 作物遺傳育種·種質資源·分子遺傳學 ? 上一篇    下一篇

棉花CRVW的克隆與抗黃萎病功能分析

王秋瑩,王偉巧,張艷,王國寧,吳立強,張桂寅,馬峙英,楊君(),王省芬()   

  1. 河北農業大學農學院/教育部華北作物種質資源研究與利用重點實驗室,河北保定071001
  • 收稿日期:2019-01-08 接受日期:2019-03-22 出版日期:2019-06-01 發布日期:2019-06-11
  • 通訊作者: 楊君,王省芬 E-mail:[email protected];[email protected]
  • 作者簡介:王秋瑩,Tel:0312-7528415;E-mail:[email protected]
  • 基金資助:
    河北省自然科學基金(C2016204098);河北省科技支撐計劃(16226307D)

Cloning and Functional Characterization of Gene CRVW Involved in Cotton Resistance to Verticillium Wilt

WANG QiuYing,WANG WeiQiao,ZHANG Yan,WANG GuoNing,WU LiQiang,ZHANG GuiYin,MA ZhiYing,YANG Jun(),WANG XingFen()   

  1. College of Agronomy, Hebei Agricultural University/North China Key Laboratory for Crop Germplasm Resources, Ministry of Education, Baoding 071001, Hebei

摘要:

目的 黃萎病(Verticillium wilt)是棉花生產上的重要病害,嚴重影響棉花的產量和品質。棉花基因組測序工作的完成為抗病基因挖掘提供了重要的信息資源。通過對一個尚未有功能注釋的陸地棉基因CRVW(cotton resistance to Verticillium wilt)進行克隆與抗病功能驗證,為棉花基因組信息完善、抗病機制解析和分子育種等方面奠定基礎。方法 根據參考基因組序列設計引物,同源克隆陸地棉(Gossypium hirsutum)農大601(ND601)中CRVW的開放讀碼框(open reading frame,ORF)。利用在線工具ProtParam預測蛋白氨基酸組成、分子量、理論等電點、不穩定指數和總平均親水性等性質;應用PSIPRED v3.3預測蛋白二級結構;在線工具ProtComp v. 9.0進行亞細胞定位預測;PlantCARE在線軟件分析順式作用元件。構建CRVW與綠色熒光蛋白基因融合表達載體,通過基因槍介導法轉化洋蔥表皮細胞,觀察CRVW的表達位置。利用qRT-PCR檢測CRVW在棉花不同組織、黃萎病菌脅迫條件下不同抗、感品種間,以及水楊酸(salicylic acid,SA)誘導處理條件下的表達模式。構建CRVW沉默載體,應用病毒誘導的基因沉默(virus-induced gene silencing,VIGS)技術進一步驗證該基因在棉花中的抗病功能。檢測CRVW沉默后一些與植物抗病調控相關標志基因的表達變化,分析其介導的抗病通路。結果 從陸地棉品種ND601中克隆到CRVW的ORF,其全長780 bp,編碼259個氨基酸殘基,分子量約為30.2 kD,理論等電點9.59;蛋白二級結構含69.50%不規則卷曲、17.76% α-螺旋、11.20%延伸鏈和1.54% β-卷曲。綜合生物信息學預測和熒光觀察結果,顯示CRVW主要存在于植物細胞膜和細胞質。CRVW在棉花根、莖和葉中都有表達,但在根中的表達量最高。CRVW的ORF上游序列(CRVW-P)中包括響應乙烯(ethylene)、SA、生長素(auxin)和脫落酸(abscisic acid)等4種激素信號的順式作用元件。另外,CRVW-P還包括一些與傷害、防御、脅迫、病菌、干旱和低溫等相關的順式作用元件。SA噴灑處理后,CRVW顯著上調表達。黃萎病菌脅迫后,CRVW在抗病品種ND601和感病品種中棉所8號(CCRI8)中均顯著上調表達,但在感病品種中上調表達的發生時間明顯滯后。黃萎病菌處理20 d后,CRVW沉默組棉苗表現出比對照(CK)組更明顯的黃化、萎蔫和落葉等黃萎病病癥。進一步統計分析顯示,CRVW沉默組病指顯著高于CK組,表明CRVW沉默顯著降低了棉苗對黃萎病菌的抗性。沉默CRVW后,棉苗中SA含量顯著降低;ICS1(isochorismate synthase 1)、EDS1(enhanced disease susceptibility 1)、PAD4(phytoalexin deficient 4)、NPR1(nonexpresser of PR gene 1)和PR1(pathogenesis-related protein 1)等與SA積累和信號調控相關的標志基因均發生顯著下調表達。結論 CRVW定位于細胞質和細胞膜,主要在棉花根部表達,可能通過SA信號通道參與棉花抗黃萎病反應過程。

關鍵詞: 棉花, 黃萎病, CRVW, 克隆, 病毒誘導的基因沉默, 抗性

Abstract:

【Objective】 Verticillium wilt is an important disease in cotton production, and it seriously affects the yield and quality of cotton. Genome sequences of Gossypium hirsutum provide valuable information resources for searching for resistance genes. In this study, an uncharacterized gene, designed as CRVW (cotton resistance to Verticillium wilt), was cloned and identified for disease resistance. The results will lay a foundation for upgrading cotton genomic information, further studying the resistance mechanism and molecular breeding. 【Method】 The open reading frame (ORF) of CRVW was cloned from upland cotton cultivar ND601 using the primers, which were designed according to the reference genome sequence. The online software ProtParam was used to predict protein properties, including amino acid composition, molecular weight, the theoretic isoelectric point, instability index and grand average of hydropathicity. PSIPRED v3.3 was used to predict the protein secondary structure. The prediction of protein subcellular localization and cis-acting elements in the promoter was performed using ProtComp v. 9.0 and PlantCARE, respectively. To elucidate the subcellular localization of the CRVW protein, the CRVW-GFP fusion construct was transformed into onion epidermal cells by particle bombardment. qRT-PCR was performed using normal cotton tissues and tissues that were treated with exogenous application of salicylic acid (SA) and Verticillium dahliae stress. The function of CRVW involving in cotton resistance to V. dahliae was further verified by the technology of virus-induced gene silencing (VIGS). To preliminarily analyze the disease resistance pathway mediated by CRVW, the expression of some marker genes related to plant disease resistance was assayed in CRVW-silenced plants.【Result】 A 780 bp ORF of CRVW was successfully cloned from G. hirsutum ND601. CRVW encodes a putative protein of 259 amino acids with a molecular mass of 30.2 kD and an isoelectric point of 9.59. The protein secondary structure of CRVW contains 69.50% random coil, 17.76% α-helical, 11.20% extension and 1.54% β-sheet. By bioinformatics prediction and fluorescence observation, we found that CRVW was mainly located in the cell membrane and cytoplasm. CRVW was expressed in the roots, stems and leaves of cotton, but the highest expression occurred in the roots. The upstream sequence of CRVW ORF (CRVW-P) contains cis-acting elements in response to four kinds of hormones, including ethylene, SA, auxin and abscisic acid. Additionally, CRVW-P includes a few other elements relating to injury, defense, stress, disease, drought and low temperature. The expression of CRVW was significantly upregulated in the leaves sprayed with SA. After inoculated with V. dahliae, CRVW was dramatically upregulated both in resistant cultivar ND601 and susceptible cultivar CCRI8, but the upregulated expression in susceptible cultivar lagged behind in the resistant cultivar. After 20 days inoculated with V. dahliae, CRVW-silenced cotton seedlings showed more clearly chlorosis, wilting and defoliating comparing to CK. Further statistical analysis showed that CRVW-silenced cotton seedlings had higher disease index than the CK, suggesting that the silence of CRVW significantly reduced the resistance of cotton seedling to V. dahliae. Endogenous SA content in CRVW-silenced cotton seedlings was significantly lower than in CK. The expression of marker genes related to SA accumulation and signal regulation, including ICS1 (isochorismate synthase 1), EDS1 (enhanced disease susceptibility 1), PAD4 (phytoalexin deficient 4), NPR1 (nonexpresser of PR gene 1) and PR1 (pathogenesis- related protein 1), were significantly down-regulated after silencing CRVW.【Conclusion】 CRVW is located in the cytoplasm and the cell membrane, mainly expressed in cotton roots, and involved in the process of cotton resistance to Verticillium wilt, perhaps through SA-mediated defense pathway.

Key words: cotton, Verticillium wilt, CRVW, clone, virus-induced gene silencing, resistance

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