高羊茅黔草1号和黔草2号LEA3基因克隆与苗期干旱胁迫表达  

Cloning of LEA3 Gene from Two Festuca arundinacea Cultivars Qiancao 1 and Qiancao 2 and Their Expression Under Drought Stress

作　　者：杨琴琴 胡江[1] 牛熙[1,2] 黄世会 冉雪琴 王嘉福[1,2] 王江岚[1] 令狐绍进 吕婷 李骋 YANG Qin-qin;HU Jiang;NIU Xi;HUANG Shi-hui;RAN Xue-qin;WANG Jia-fu;WANG Jiang-lan;LINGHU Shao-jin;LYU Ting;LI Cheng(Institute of Agro-Bioengineering/College of Life Sciences,Guizhou University,Guizhou Key Laboratory of Agro-Bioengineering/The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guizhou Guiy-ang 550025,China;The 2011 Collaboration Innovation Center for Mountain Ecology and Agro-Bioengineering in Guizhou Province,Guizhou University,Guizhou Guiyang 550025,China;College of Animal Sciences,Guizhou University,Guizhou Guiyang 550025,China)

机构地区：[1]贵州大学农业生物工程研究院/生命科学学院,贵州省农业生物工程重点实验室/山地植物资源保护与种质创新省部共建教育部重点实验室,贵州贵阳550025 [2]贵州省山地生态与农业生物工程2011协同创新中心,贵州贵阳550025 [3]贵州大学动物科学学院,贵州贵阳550025

出　　处：《西南农业学报》2020年第12期2736-2744,共9页Southwest China Journal of Agricultural Sciences

基　　金：山地植物资源保护与种质创新省部共建教育部重点实验室开放课题基金项目“高羊茅DREB2与LEA3基因的抗旱机制与功能研究”(MOELP-201707);大学生创新创业训练计划项目“高羊茅LEA3基因的克隆及其抗旱性的研究”(201710657074)。

摘　　要：【目的】克隆贵州本地高羊茅品种黔草1号和黔草2号的第三组晚期胚胎发生丰富蛋白(Late Embryogenesis Abundant Protein,Group 3,LEA3)基因,分析其生物信息学及其苗期干旱胁迫的表达量变化,为进一步探讨LEA3基因的功能和高羊茅抗旱分子机制提供理论基础。【方法】参照NCBI已登录大麦LEA3基因序列,利用RT-PCR获得黔草1号和黔草2号LEA3基因cDNA序列,应用相关软件对氨基酸序列进行分析,采用实时荧光定量PCR(qRT-PCR)分析苗期干旱胁迫的表达量。【结果】黔草1号和黔草2号LEA3基因完整的ORF框长度分别为609和642 bp,分别编码203个氨基酸和213个氨基酸;黔草1号LEA3蛋白比黔草2号缺失1个基元序列(包含11个氨基酸),且两者LEA3基因编码的氨基酸序列存在12个位点的氨基酸差异;黔草1号LEA3基因编码蛋白分子量为20.72 kDa,等电点8.55,总平均亲水性为-0.971;黔草2号LEA3基因编码蛋白分子量为22.01 kDa,等电点8.99,总平均亲水性为-1.069;两者的LEA3蛋白均没有跨膜结构,且均以α-螺旋结构占主导,与大麦、山羊草、冰草和小麦的LEA3具有高度的同源性,与小麦、旱麦草、大麦、山羊草、无芒雀麦、冰草的LEA3蛋白聚为一类;苗期干旱胁迫下黔草1号LEA3基因的表达量在整个胁迫过程中总体高于黔草2号,2个品种高羊茅在-2.5 MPa PEG 6000胁迫下的LEA3基因表达量高于-1.5 MPa PEG 6000胁迫。【结论】黔草1号和黔草2号的抗旱性与LEA3基因的表达量呈正相关,相同干旱胁迫条件下,黔草1号LEA3的表达量高于黔草2号,推测在受到干旱胁迫时,黔草1号更能适应干旱环境。【Objective】 The aim of this paper was to explore the function of LEA3 gene and the molecular mechanism of drought resistance of Festuca Arundinacea, for which LEA3 gene from two Guizhou native F. Arundinacea cultivars(Qiancao 1 and Qiancao 2) were cloned and the bioinformatics of LEA3 genes and their expression under drought stress at seedling stage were analyzed as well. 【Method】In this experiment, we cloned LEA3 genes of Qiancao 1 and Qiancao 2 by RT-PCR, referring to the sequences recorded by NCBI. At the same time, the amino acid sequence was analyzed by relevant software, and the expression of amino acid under drought stress was analyzed by real-time fluorescence quantitative PCR(qRT-PCR). 【Result】In this study, we found that LEA3 genes in Qiancao 1 and Qiancao 2 consisted of open reading frame(ORF) of 609 bp and 642 bp respectively, which encoded proteins of 203 and 213 amino acids residues, respectively. Compared with Qiancao 2, the LEA3 protein of Qiancao 1 lacks the motif composed of 11 amino acids. At the same time, 11 amino acid differences were found between Qiancao 1 and Qiancao 2. Bioinformatics analysis showed that the molecular weight of Qiancao 1 LEA3 protein was 20.72 kDa, the isoelectric point was 8.55, and the grand average of hydropathicity(GRAVY) was-0.971. The LEA3 protein of Qiancao 2 has a molecular weight of 22.01 kDa, an isoelectric point of 8.99, and a GRAVY of-1.069. The hydrophobicity analysis showed that both Qiancao 1 and Qiancao 2 LEA3 proteins had no transmembrane structure, and the hydrophilicity of Qiancao 2 LEA3 was stronger than that of Qiancao 1 LEA3. Alpha-helixes are dominant in Qiancao 1 and Qiancao 2 LEA3 proteins. LEA3 proteins of Qiancao 1 and Qiancao 2 have high homology with that of barley, goatgrass, wheatgrass and wheat. The phylogenetic tree showed that LEA3 proteins of Qiancao 1, Qiancao 2, wheat, ryegrass, barley, goatgrass, bromus inermis and wheatgrass were clustered into one group. The results of qRT-PCR analysis showed that the expression of Qian

关 键 词：高羊茅 LEA3基因 基因克隆 干旱 表达分析 

分 类 号：S816.51[农业科学—饲料科学]