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棉花学报 ›› 2015, Vol. 27 ›› Issue (2): 111-117.doi: 10.11963/issn.1002-7807.201502003

• 研究与进展 • 上一篇    下一篇

棉花GhSKIP35基因的克隆与表达分析

刘凯,张华崇,齐放军,张文蔚*,简桂良*   

  1. 中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京  100193
  • 收稿日期:2014-09-15 出版日期:2015-03-15 发布日期:2015-03-15
  • 通讯作者: zwwei@sohu.com,gljian@ippcaas.cn
  • 作者简介:刘凯(1988―)男,硕士,kerry0726@126.com
  • 基金资助:

    公益性行业(农业)科研专项(201503109)

Cloning and Expression Analysis of GhSKIP35 Gene in Gossypium hirsutum L.

Liu kai, Zhang Huachong, Qi Fangjun, Zhang Wenwei*, Jian Guiliang*   

  1. Institute of Plant Protection, CAAS, the State Key Laboratory for Biology of Plant Diseases and Insect Pest, Beijing 100193, China
  • Received:2014-09-15 Online:2015-03-15 Published:2015-03-15

摘要: 以中植棉KV1 SSH文库为基础,从陆地棉中植棉KV1中克隆出来泛素途径SKIP35基因,命名为GhSKIP35。该基因的开放阅读框全长为1863个核苷酸,编码620个氨基酸,含有1个ANK保守结构域。进化分析表明,GhSKIP35蛋白与甜橙(Citrus sinensis)的SKIP35蛋白相似性最高。qRT-PCR分析显示:在接种大丽轮枝菌V991菌系后初期GhSKIP35基因表达量激增,12 h便可达到顶峰,24~48 h逐渐下降,推测它在陆地棉抗黄萎病早期防卫反应中起重要作用。利用VIGS(Virus induced gene silencing)技术,在中植棉KV1中成功地沉默GhSKIP35基因;对沉默棉株接种大丽轮枝菌V991鉴定显示,其病情指数为50.75,而野生型的中植棉KV1和转化空载体棉株的病情指数分别为9.38和11.54。GhSKIP35基因沉默后,中植棉KV1对黄萎病的抗性丧失,表明GhSKIP35基因在陆地棉抗黄萎病的过程中起重要作用。

关键词: 棉花; 大丽轮枝菌; SKIP35; qRT-PCR; VIGS

Abstract: A new gene, GhSKIP35, was cloned from cotton cultivar Zhongzhimian KV1, which was selected by screening a suppression subtractive hybridization library of Zhongzhimian KV1. The open reading frame (ORF) was 1863 nucleotides (nts) long and encoded a protein of 620 amino acids. The putative protein contains a conserved ANK domain. The cloned gene was most closely related to Citrus sinensis SKIP35 protein by phylogenetic analysis. Real-time quantitative reverse transcription polymerase chain reaction showed that the GhSKIP35 gene was rapidly activated after inoculation with Verticillium dahliae. Gene expression reached a peak at 12 h post-inoculation (hpi), and then its expression gradually decreased within 24―48 h. It was assumed that GhSKIP35 might play an important role in the defense reaction against Verticillium wilt. Virus induced gene silencing was used to silence endogenous genes in resistant upland cotton cultivar Zhongzhimian KV1 by targeting a fragment of GhSKIP35. V991 was used to inoculate silenced cotton plants to identify disease resistance. The results showed that the disease indices of wild type Zhongzhimian KV1 and vector control plants were 9.38 and 11.54, respectively. The disease index of silenced GhSKIP35 plants was 50.75. These results confirmed that the GhSKIP35 gene was related to Verticillium resistance in cotton.

Key words: cotton; Verticillium dahliae; SKIP35; real-time quantitative reverse-transcription polymerase chain reaction; virus induced gene silencing

中图分类号: 
  • S435.621