甘蔗CIPK基因的同源克隆与表达

Cloning and Expression Analysis of CIPK Gene in Sugarcane

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。

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 ofCIPK15gene fromZea mays, a full-length cDNA se-quence of serine/threonine kinase gene termed asScCIPK was cloned by RT-PCR method from sugarcane （Saccharum Complex）. The sequence analysis showed thatScCIPK 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 theScCIPK 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 wasα-helix. Real-time quantitative PCR（RT-qPCR） analysis revealed that the expression ofScCIPKwas higher in bud than in other tissues, meanwhile the inducible expression level ofScCIPK was most significantly up-regulated under the ABA stress, 5.3 times higher than that of control, which suggested thatScCIPK 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 ofScCIPKin sugarcane and promote the use of ScCIPK gene in sugarcane genetic engineering.