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作物学报 ›› 2015, Vol. 41 ›› Issue (03): 499-506.

• 研究简报 • 上一篇    下一篇

甘蔗CIPK基因的同源克隆与表达

黄珑，苏炜华，张玉叶，黄宁，凌辉，肖新换，阙友雄，陈如凯   

  1. 福建农林大学 / 农业部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗产业技术研发中心, 福建福州 350002
  • 收稿日期:2014-07-14 修回日期:2014-12-09 出版日期:2015-03-12 网络出版日期:2014-12-30
  • 通讯作者: 阙友雄, E-mail: queyouxiong@126.com; 陈如凯, fafu948@126.com
  • 基金资助:

    本研究由国家自然科学基金项目(31340060), 福建省高等学校新世纪优秀人才支持计划项目(JA14095)和国家现代农业产业技术体系建设专项(CARS-20)资助。

Cloning and Expression Analysis of CIPK Gene in Sugarcane

HUANG Long,SU Wei-Hua,ZHANG Yu-Ye,HUANG Ning,LING Hui,XIAO Xin-Huan,QUE You-Xiong*,CHEN Ru-Kai*   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University / Sugarcane Research & Development Center, China Agricultural Technology System, Fuzhou 350002, China
  • Received:2014-07-14 Revised:2014-12-09 Published:2015-03-12 Published online:2014-12-30
  • Contact: 阙友雄, E-mail: queyouxiong@126.com; 陈如凯, fafu948@126.com

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摘要：

CIPK (calcineurin B-like-interacting protein kinase)是植物特有一类的丝氨酸/苏氨酸蛋白激酶，该蛋白在植物响应逆境胁迫中发挥着重要的作用，尤其与非生物逆境胁迫(干旱、高盐、ABA等)的信号传导密切相关。根据玉米CIPK15基因(EU957447.1, 2247 bp)核酸序列保守区域设计1对同源克隆PCR引物，以甘蔗品种崖城05-179的cDNA为模板，通过RT-PCR扩增得到甘蔗CIPK基因的一条全长cDNA序列(GenBank登录号为KM114052)。序列分析结果表明，甘蔗ScCIPK基因全长1782 bp，具有完整的开放阅读框(ORF, 91~1631 bp)，编码513个氨基酸，该基因具有CIPK基因的2个特征结构域(Kc-like superfamily和AMPKA-C-like superfamily)。生物信息学分析显示该基因编码的蛋白定位于内质网，为可溶性蛋白，不存在信号肽，二级结构元件多为α-螺旋，含有多个保守功能域，主要参与中间代谢。实时定量PCR表达分析表明, 该基因表达具有组织特异性，虽在甘蔗各组织中均有表达，但在芽中的表达量最高。该基因在PEG、NaCl、ABA、SA和MeJA的胁迫诱导过程中，受ABA胁迫后表达量最高，约为对照的5.3倍，推测该基因的表达与甘蔗抗干旱和抗渗透胁迫有关。

关键词: 甘蔗, CIPK 基因, 同源克隆, 生物信息学, 实时定量PCR

Abstract:

CIPK (calcineurin B-like-interacting protein kinase) is a plant specific class of serine / threonine protein kinases, which plays an important role in plant response to stress, especially relates with the signal transduction for biotic stresses (drought, high salt, ABA). According to the primers designed on the conserved domain of CIPK15 gene from Zea mays, a full-length cDNA sequence of serine/threonine kinase gene termed as ScCIPK was cloned by RT-PCR method from sugarcane (Saccharum Complex). The sequence analysis showed that ScCIPK had a length of 1782 bp containing the open reading frame (ORF, 91–1631 bp), which encoded 513 amino acids residues with two conserved domains (Kc-like superfamily and AMPKA-C-like superfamily). The characters predicted based on the bioinformatics analysis revealed that the ScCIPK gene of sugarcane was a soluble acidic protein, which has two conserved functional domains with the main function for central_intermediary_metabolism, and its protein was located in endoplasmic reticulum (membrane). The mainly secondary structure element was α-helix. Real-time quantitative PCR(RT-qPCR) analysis revealed that the expression of ScCIPK was higher in bud than in other tissues, meanwhile the inducible expression level of ScCIPK was most significantly up-regulated under the ABA stress, 5.3 times higher than that of control, which suggested that ScCIPK most probably involves in sugarcane resistance to drought and osmotic stresses. The results in this study could provide a basis of cloning and functional identification of other members of ScCIPK in sugarcane and promote the use of ScCIPK gene in sugarcane genetic engineering.

Key words: Sugarcane, CIPK, homology cloning, Bioinformatics, Real-time PCR

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