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作物学报 ›› 2016, Vol. 42 ›› Issue (12): 1779-1786.doi: 10.3724/SP.J.1006.2016.01779

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一个受黄萎病菌诱导的海岛棉功能基因GbVWR的克隆与表达

张力佳**,张艳**,荣伟,杨君,张桂寅,吴立强,李志坤,吴金华,马峙英,王省芬*   

  1. 华北作物种质资源研究与利用重点实验室 / 河北省作物种质资源重点实验室 / 河北农业大学,河北保定 071001
  • 收稿日期:2016-03-14 修回日期:2016-06-20 出版日期:2016-12-12 网络出版日期:2016-06-27
  • 通讯作者: 王省芬, E-mail: cotton@hebau.edu.cn
  • 基金资助:

    本研究由河北省自然科学基金项目(C2013204141), 高等学校博士学科点专项科研基金(20131302120002)和河北省百名优秀创新人才支持计划(14226308D)资助。

Cloning and Expression Analysis of a Functional Gene GbVWR Induced by Verticillium dahliae in Gossypium barbadense

ZHANG Li-Jia**,ZHANG Yan**,RONG Wei,YANG Jun,ZHANG Gui-Yin,WU Li-Qiang,LI Zhi-Kun,WU Jin-Hua,MA Zhi-Ying,WANG Xing-Fen*   

  1. North China Key Laboratory for Crop Germplasm Resources of Education Ministry / Key Laboratory for Crop Germplasm Resources of Hebei / Agricultural University of Hebei, Baoding 071001, China
  • Received:2016-03-14 Revised:2016-06-20 Published:2016-12-12 Published online:2016-06-27
  • Contact: Wang Shengfen, E-mail: cotton@hebau.edu.cn
  • Supported by:

    This study was supported by the Natural Science Foundation of Hebei Province (C2013204141), the Special Research Found for the Doctoral Program of Higher Education (20131302120002), and the Science and Technology Support Project of Hebei Province (14226308D).

摘要:

黄萎病是一种土传真菌维管束病害,严重影响棉花产量和品质。挖掘抗黄萎病相关基因对于棉花抗黄萎病遗传改良具有重要意义。本研究通过筛选黄萎病菌胁迫下的海岛棉全长cDNA文库和陆地棉SSH文库,获得一个与黄萎病菌胁迫相关的基因,命名为GbVWR。生物信息学特征和基因表达分析表明,GbVWR基因全长520 bp,开放读码框198 bp,编码65个氨基酸残基组成的蛋白,理论等电点为5.32,包含一个信号肽和一个跨膜区,是一种分泌蛋白。GbVWR基因可以诱导表达7 kD的蛋白。在GbVWR基因起始密码子上游1500 bp的核苷酸序列区间预测到真菌激发子响应、激素响应、伤口响应及黄酮生物合成基因调节等应答元件。GbVWR基因在海岛棉根中表达最高,茎中其次,叶中最低。受黄萎病菌诱导后,GbVWR基因在接菌后2 h可对黄萎病菌作出应答反应。此外SA、ET和GA均可显著诱导GbVWR基因的表达。初步推断GbVWR是海岛棉抵御黄萎病菌过程中一个新的功能基因,通过参与多种激素信号途径发挥功能

关键词: 海岛棉, 抗黄萎病, 未知基因(GbVWR), 克隆, 基因表达

Abstract:

Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes severe losses in cotton yield and fiber quality. Mining functional genes related to resistance against V. dahliae will benefit efforts to genetically improve crop plants. In this study, we identified a gene that involved in cotton defense against V. dahliae based on screening the full-length cDNA library and suppression subtractive hybridization library (SSH) induced by V. dahliae in Gossypium barbadense and Gossypium. hirsutum, respectively. Sequence analysis indicated that there was no any annotation in NCBI database, and we named the sequence from G. barbadense as GbVWR. We characterized GbVWR gene and analyzed its expression. The full length cDNA of GbVWR was 520 bp including a 198 bp open reading frame (ORF), encoding 65 amino acid residues. Bioinformatic analyses suggested that GbVWR belonged to secretory protein and tis theoretical isoelectric point was 5.32. Using pET-32a(+) as a fused expression vector, a recombinant plasmid pET32a-GbVWR was constructed. The recombinant protein was induced in Escherichia coli BL21 (DE3) with 1.0 mmol L–1 IPTG then GbVWR could express about 7 kD protein in E. coli BL21 (DE3). In addition, diverse cis-acting promoter elements involved in fungal elicitor response, hormone response, wound-response, and flavonoid biosynthetic gene regulation were discovered in the promoter region of GbVWR. qPCR analysis showed that expression level of GbVWR was the highest in roots, and was significantly induced by V. dahliae. Besides, GbVWR could also be induced by SA, ET and GA treatments, respectively. In conclusion, GbVWR is a new functional gene, which involved in multiple signal pathways in cotton defense response to Verticillium wilt.

Key words: Gossypium barbadense, Verticillium wilt resistance, GbVWR, Clone, Gene expression

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