菊芋抗逆性评估及HtCPI基因的克隆与表达分析  

Cloning and expression analysis of HtCPI gene with the evaluation of the stress resistance in Helianthus tuberosus

作　　者：刘鹏[1] 郦枫 曹林[1] 吴玉环[2] 章艺[3] 徐根娣[1] 马丽[1] 刘丹[1] 

机构地区：[1]浙江师范大学化学与生命科学学院,浙江金华321004 [2]杭州师范大学生命与环境科学学院,浙江杭州310036 [3]浙江旅游职业学院,浙江杭州311231

出　　处：《浙江师范大学学报（自然科学版）》2017年第2期188-195,共8页Journal of Zhejiang Normal University:Natural Sciences

基　　金：国家自然科学基金资助项目(41571049;41461010);浙江省自然科学基金资助项目(5100390)

摘　　要：在逆境胁迫测试的基础上,利用RT-PCR技术从菊芋叶片中克隆了半胱氨酸蛋白酶抑制剂基因的完全开放阅读框序列,并进行了生物信息学和胁迫表达分析.结果表明:获得的基因序列具有半胱氨酸蛋白酶抑制剂特征性的结构域,HtCPI由333 bp的核苷酸组成;蛋白质结构预测,HtCPI推定的HtCY蛋白具有跨膜结构和信号肽;系统进化树分析,HtCY与山杨的CY蛋白相似性为96%,与姜黄的CY相似性为94%;经氨基酸序列比对,推断该蛋白有一个高度保守的QKVKQ结构域和一个保守的LAKFAVDHHN结构域;蛋白质亚细胞定位结果表明,HtCY可能位于内质网上.组织特异性检测表明,HtCPI在菊芋根、茎、叶中均有表达,在茎中的表达量最高.实时定量荧光PCR分析表明,水杨酸、氯化钠、氯化铝和聚乙二醇等处理均能诱导菊芋根和叶中HtCPI基因表达上调;荧光定量分析显示,虽然在不同的胁迫因子条件下其上调的程度有一定的差异,但均在4 h后表达量发生明显的上升,即对水杨酸、氯化钠、氯化铝和聚乙二醇等处理均存在转录响应.推测该基因参与了菊芋对生物或非生物胁迫的防御机制.On the basis of the stress test, the open reading frame of CPI （ Cysteine proteinase inhibitor ） gene was cloned from Helianthus tuberosus leafs with RT-PCR, for bioinformaties and stress expression analysis. The results showed that the sequence obtained had a protein domain with the characteristics of CPI, contributed by 333 bp nucleotides. Prediction of protein structure indicated that, HtCY protein encoded by HtCPI had trans membrane domain and signal peptide. Phylogenetic analysis demonstrated that, HtCY had similarity of 96% with CY of aspen, and had similarity of 94% with CY of turmeric; by the comparison between sequences of the amino acids, it was inferred that the protein had a highly conserved domain named QKVKQ and a conser- vative domain named LAKFAVDHHN. POST results showed that, HtCY might be located in the endoplasmic reticulum. Tissue-specific analysis showed that, HtCPI could be found in roots, stems, leaves, which had the highest expression in the stem. Real-Time PCR analysis showed that salicylic acid （SA） , sodium chloride （ NaC1 ）, aluminum chloride （ A1C13 ）, polyethylene glycol （ PEG} and other treatments could improve the expression of HtCPI regulated gene from Helianthus tuberosus root and leaves, fluorescence quantitative analysis showed that although there were some differences between different groups on the degree, but all of them increased significantly after 4 hours. SA, NaC1, A1C13 , PEG and other treatments could motivate the transcriptional response of the gene, suggesting that the gene involved in the defense of Helianthus tuberosus to biotic and abiotic stress, for the foundation of further study of CPI in plant physiological functions.

关 键 词：菊芋 胁迫 半胱氨酸蛋白酶抑制剂 克隆 表达 基因 

分 类 号：S567.23[农业科学—中草药栽培]