中國農業科學 ?? 2019, Vol. 52 ?? Issue (12): 2021-2032.doi: 10.3864/j.issn.0578-1752.2019.12.001

? 作物遺傳育種·種質資源·分子遺傳學 ? 上一篇    下一篇

水稻轉錄因子OsWRKY68蛋白質的表達特征及其功能特性

陳悅,王田幸子,楊爍,張彤,馬金姣,燕高偉,劉玉晴,周艷,史佳楠,蘭金蘋,魏健,竇世娟,劉麗娟,楊明,李莉云,劉國振()   

  1. 河北農業大學生命科學學院,河北保定 071001
  • 收稿日期:2019-01-30 接受日期:2019-03-28 出版日期:2019-06-16 發布日期:2019-06-22
  • 通訊作者: 劉國振 E-mail:[email protected]
  • 作者簡介:陳悅,E-mail: [email protected]
  • 基金資助:
    國家自然科學基金(31400700);國家自然科學基金(31171528)

Expression Profiling and Functional Characterization of Rice Transcription Factor OsWRKY68

Yue CHEN,TianXingZi WANG,Shuo YANG,Tong ZHANG,JinJiao MA,GaoWei YAN,YuQing LIU,Yan ZHOU,JiaNan SHI,JinPing LAN,Jian WEI,ShiJuan DOU,LiJuan LIU,Ming YANG,LiYun LI,GuoZhen LIU()   

  1. College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei
  • Received:2019-01-30 Accepted:2019-03-28 Online:2019-06-16 Published:2019-06-22
  • Contact: GuoZhen LIU E-mail:[email protected]

摘要:

【目的】 水稻中有近百個WRKY轉錄因子家族成員,其中很多與生長發育、生物與非生物逆境脅迫應答有關。河北農業大學生命科學學院分子生物學與生物信息學實驗室(molecular biology & bioinformatics lab,MBB)前期發現OsWRKY68在水稻接種白葉枯病菌后誘導表達,本研究試圖進一步探究OsWRKY68的功能。【方法】 采集水稻TP309不同生長發育時期的組織樣品,包括萌發期、幼苗期、分蘗期、孕穗期和開花期的根、莖、葉、葉鞘、葉枕、穗子、花藥、穎殼和種子,以及非生物脅迫(4℃、44℃、48℃、淹、NaCl、PEG、恒光和恒暗)和激素處理(脫落酸、茉莉酸甲酯、水楊酸和乙烯利)的葉片,提取總蛋白質,用水稻OsWRKY68蛋白質特異抗體,通過免疫印跡(western blot,WB)技術系統調查OsWRKY68蛋白質在水稻正常發育過程中不同時期、不同組織、非生物脅迫及激素處理條件下的表達特征。構建RNAi載體,通過農桿菌介導的方法轉化水稻TP309,對轉基因植株進行PCR和WB鑒定,觀察OsWRKY68 RNAi轉基因植株的表型并測量其株高、分蘗數、穗長、小穗數和結實率等性狀。【結果】 調查OsWRKY68蛋白質的表達豐度,發現OsWRKY68蛋白質在水稻正常生長發育過程中基本呈組成型表達,且在大部分組織中表達豐度差異倍數不大,但在開花期的花藥中OsWRKY68表達量高于成熟穗、穗軸和穎殼;在分蘗期和孕穗期的葉鞘中不表達,僅在開花期的葉鞘中表達;在孕穗期的幼穗中,隨著幼穗長度的增加其表達豐度逐漸降低。調查水稻非生物脅迫和激素處理后OsWRKY68蛋白質的表達特征,發現鹽脅迫后OsWRKY68蛋白質的表達豐度隨時間延長持續下降,恒光處理后OsWRKY68蛋白質表達量持續上升,3 d時明顯出現一條分子質量較大的條帶(記作OsWRKY68 +),且表達量逐漸增加,茉莉酸甲酯(methyl jasmonate,MeJA)和乙烯利(ethephon,ET)處理后同樣也呈現OsWRKY68 +蛋白質豐度的增加。對轉基因植株進行鑒定和自交繁殖,對T3代的4個OsWRKY68 RNAi轉基因株系(Y316、Y317、Y326和Y337)進行PCR和WB鑒定,均表現陽性。在轉基因植株中,OsWRKY68蛋白質的表達量均低于野生型TP309。對轉基因植株進行表型觀察和性狀測量,發現與野生型相比轉基因植株的株高降低、分蘗數和結實率下降等。 【結論】 OsWRKY68蛋白質在水稻正常生長發育中發揮作用,敲低OsWRKY68蛋白質的表達能影響水稻的正常生長。OsWRKY68蛋白質可能參與鹽、光照、茉莉酸甲酯和乙烯介導的信號轉導過程。

關鍵詞: 水稻, WRKY轉錄因子, 表達特征, 免疫印跡, RNA干擾

Abstract:

【Objective】 There are nearly 100 WRKY transcription factor members in rice genome, many of them are involved in plant growth and development, biotic and abiotic stress responses. Molecular biology & bioinformatics lab identified that the expression of OsWRKY68 protein was induced after inoculation with Xanthomonas oryzae pv. oryzae (Xoo) in rice. The aim of this study is attempt to further explore the function of OsWRKY68. 【Method】Rice TP309 samples of different tissues at different developmental stages, including germination, seedling, tillering, booting and flowering stages of root, stem, leaf, sheath, cushion, panicle, anther, husk, seed, abiotic stress (4℃, 44℃, 48℃, submerge, NaCl, PEG, constant light, constant dark) and hormone treatments (abscisic acid, methyl jasmonate, salicylic acid, ethephon) were collected. Total protein were extracted and analyzed by Western blot (WB) systematically using OsWRKY68-specific antibody. The expression patterns of OsWRKY68 protein isolated from different tissues at different developmental stages, and tissues obtained from abiotic stresses and hormone treatments were investigated. RNA interfering vector was constructed and transformed to wildtype TP309 rice variety via Agrobacterium tumefaciens strategy. Identification of transgenic plants were carried out by PCR and WB. The phenotype of OsWRKY68 RNAi transgenic plants were monitored and plant height, tiller number, spike length, spikelet number and seed-setting rate were measured.【Result】By comparing the abundance of OsWRKY68 protein in different tissues, it was found that OsWRKY68 protein was expressed in a constitutive way during the normal growth and development of rice, the abundance of OsWRKY68 protein expressed among different tissues were not varied too much. However, different levels of OsWRKY68 were observed. The expression level of OsWRKY68 in anthers at flowering stage was higher than that in mature panicles, panicle axis and husk. It was not expressed in sheaths at tillering and booting stages, but it was expressed in sheaths at flowering stage. In panicles, the abundance of OsWRKY68 was decreased gradually along with the growth of the young panicle. By investigating the expression patterns of OsWRKY68 protein under abiotic stress and hormone treatments, it was found that the abundance of OsWRKY68 protein decreased steadily under salt stress. The expression of OsWRKY68 protein increased steadily at constant light treatment, a specific band (designated as OsWRKY68 +) with higher molecular weight appeared at three days and enhanced in the following timepoints. After methyl jasmonate (MeJA) and ethephon (ET) treatments, OsWRKY68 + band appeared also and its intensity increased as the treatments continues. Four homozygous OsWRKY68 RNAi transgenic lines (Y316, Y317, Y326 and Y337) were checked by PCR and WB analyses and verified at T3 generation. The abundance of OsWRKY68 protein in RNAi transgenic plants was lower than that in wildtype TP309. Phenotypic investigation revealed significant reduction in plant height, tiller number and seed setting rate in transgenic plants.【Conclusion】Rice OsWRKY68 protein plays an important role in the process of normal growth and development of rice. Knocking down the abundance of OsWRKY68 protein via RNAi affected the normal growth of rice. In addition, the data of expression patterns suggested that the function of OsWRKY68 protein may be involved with salt stress, light, MeJA and ethene-mediated signal transduction pathways.

Key words: rice, WRKY transcription factor, expression patterns, western blot, RNA interference

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