红花脂质转运蛋白基因的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.07.022

生物技术通报 ›› 2016, Vol. 32 ›› Issue (7): 146-151.doi: 10.13560/j.cnki.biotech.bull.1985.2016.07.022

红花脂质转运蛋白基因的克隆及表达分析

韩怡来¹, 崔琪¹, 武云云¹, 顾天瑶¹, 滕孝花³, 胡人阁¹, 吴贯达¹, 官丽莉^{1, 2}, 李海燕^{1, 2}, 李校堃²

1. 吉林农业大学生命科学学院,长春 130118；
2. 吉林农业大学生物反应器与药物开发教育部工程研究中心,长春 130118；
3. 通化市第一中学,通化 134000

收稿日期:2015-07-22 出版日期:2016-07-25 发布日期:2016-07-25
作者简介:韩怡来,男,研究方向：生物技术;E-mail：994435840@qq.com
基金资助:
国家高技术研究发展计划（“863”）项目（2011AA100606）,吉林省教育厅“十三五”科学技术研究项目重点项目（吉教科合字[2016]179号） ,国家自然科学基金资助项目（31201237）,吉林农业大学大学生创新创业项目（201610193046,201410193045）,吉林省科技厅中青年领军人才及优秀创新团队项目（20111815）,教育部博士点基金-青年教师基金项目（20122223120002）

Cloning and Expression Analysis of Lipid Transfer Protein LTP Gene in Carthamus tinctorius

HAN Yi-lai¹, CUI Qi¹, WU Yun-yun¹, GU Tian-yao¹, TENG Xiao-hua³, HU Ren-ge¹, WU Guan-da¹, GUAN Li-li^{1, 2}, LI Hai-yan^{1, 2}, LI Xiao-kun²

1. College of Life Sciences,Jilin Agricultural University,Changchun 130118;
2. Ministry of Education Bioreactor and Drug Development Research Center,Jilin Agricultural University,Changchun 130118;
3. Tonghua No.1 Senior High School,Tonghua 134000

Received:2015-07-22 Published:2016-07-25 Online:2016-07-25

摘要/Abstract

摘要： 克隆红花脂质转运蛋白基因（Lipid transfer protein,LTP）,并进行生物信息学及表达分析,旨为研究LTP在红花抵抗逆境胁迫中的作用提供依据。通过RT-PCR方法从红花种子中克隆LTP基因序列,通过生物信息学对该基因蛋白的特征进行分析,构建LTP与相关物种LTP的系统进化树,利用Real-time PCR方法分析在红花不同号组织中LTP基因的表达量。结果显示,LTP基因ORF全长294 bp,编码97个氨基酸,相对分子量为7.46 kD,等电点为8.91。红花LTP蛋白包含一个长为29个氨基酸残基的信号肽序列;该蛋白含有一个丝氨酸磷酸化位点;三级结构预测表明该蛋白是由3个α-螺旋和一个β-转角简单的缠绕在无规则卷曲上的简单结构。分子进化表明,红花LTP基因与十字花科的甘蓝型油菜进化关系最近。通过荧光定量PCR对红花LTP基因的组织表达特异性进行分析,结果表明CtLTP在不同组织的表达水平具有显著差异,在种子和花中呈现高表达,而在其他组织中低表达。

关键词: 脂质转运蛋白, 红花, 荧光定量PCR, 生物信息学分析

Abstract: LTP（Lipid transfer protein）gene was cloned,its bioinformatics and expression were analyzed,providing the basis for the study the role of LTP gene in safflower resisting stresses. The sequence of LTP gene was cloned by RT-PCR technique,the protein characteristics were analyzed using bioinformatics,and the phylogenetic tree of LTP for safflower and other species was constructed. The expressions of LTP gene in the different tissues were analyzed using Real time-PCR. Sequence analysis showed that ORF of LTP was 294 bp,encoding a protein of 97 amino acids with a molecular weight of 7.46 kD,isoelectric point of 8.91. Safflower LTP protein contained a long signal peptide sequence of 29 amino acid residues,and the protein contained a serine phosphorylation site. Tertiary structure prediction indicated that the protein was a simple structure of 3 α-helix and 1 β-turn twining in random coils. Molecular evolution showed that the evolutionary relationship of LTP between Brassica of cruciferous and safflower was the most similar. Tissue-specific expression analysis of safflower LTP by fluorescence quantitative PCR showed that the gene expression levels in different tissues presented a significant difference,while high expression in seeds and flowers,and low expression in other tissues.

Key words: lipid transfer protein, safflower, Real-time PCR, bioinformatics analysis

韩怡来, 崔琪, 武云云, 顾天瑶, 滕孝花, 胡人阁, 吴贯达, 官丽莉, 李海燕, 李校堃. 红花脂质转运蛋白基因的克隆及表达分析[J]. 生物技术通报, 2016, 32(7): 146-151.

HAN Yi-lai, CUI Qi, WU Yun-yun, GU Tian-yao, TENG Xiao-hua, HU Ren-ge, WU Guan-da, GUAN Li-li, LI Hai-yan, LI Xiao-kun. Cloning and Expression Analysis of Lipid Transfer Protein LTP Gene in Carthamus tinctorius[J]. Biotechnology Bulletin, 2016, 32(7): 146-151.

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红花脂质转运蛋白基因的克隆及表达分析

Cloning and Expression Analysis of Lipid Transfer Protein LTP Gene in Carthamus tinctorius

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