高羊茅Fa6-SFT基因克隆、亚细胞定位及表达分析  

Cloning, subcellular localization and expression analysis of Fa6-SFT gene in tall fescue

作　　者：陈锡[1,2,3] 赵德刚 陈莹[1] 刘晓霞[1] 袁旸旸 王小利 CHEN Xi;ZHAO Degang;CHEN Ying;LIU Xiaoxia;YUAN Yangyang;WANG Xiaoli(Guizhou Institute of Prataculture,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China;Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region,Ministry of Education,Guiyang 550025,China;National-Local Joint Engineering Research Center of Karst Region Plant Resources Utilization&Breeding of Guizhou Province,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州省农业科学院草业研究所,贵阳550006 [2]山地植物资源保护与种质创新教育部重点实验室,贵阳550025 [3]贵州大学喀斯特山区植物资源利用与育种国家地方联合工程研究中心,贵阳550025

出　　处：《植物生理学报》2020年第4期759-770,共12页Plant Physiology Journal

基　　金：贵州省农业科学院青年基金([2018]80);贵州省科技平台及人才团队专项资金项目([2016]5634);贵州省科技计划项目([2019]1302);教育部重点实验室开放基金(MOELP-201702);贵州省农业科学院自主创新专项([2014]001)。

摘　　要：蔗糖:果聚糖-6-果糖基转移酶(sucrose:fructan 6-fructosyl-transferase, 6-SFT)是禾本科植物中果聚糖合成的关键酶,在植物抵御干旱、低温、盐胁及氮胁等非生物逆境胁迫中起着十分重要的作用。为了探讨‘黔草1号’高羊茅(Festuca arundinacea)中6-SFT基因的分子机制,利用RT-PCR结合RACE技术(rapid amplification of cDNA ends)从高羊茅中克隆了6-SFT基因。序列分析显示, cDNA全长为2 375 bp,包含一个完整的开放阅读框1 860 bp,编码蛋白含有620个氨基酸,命名为Fa6-SFT。理化性质分析显示,其蛋白分子质量为68.20 kDa,理论等电点pI为5.61,属于酸性蛋白。保守结构域分析发现,由该基因推导的氨基酸序列具有糖基水解酶32 (glycosyl hydrolase family 32, GH32)家族保守结构域。蛋白多重序列比对及进化树分析发现,高羊茅Fa6-SFT与猫尾草、黑麦草和毒麦亲缘关系最近。亚细胞定位显示Fa6-SFT主要定位于细胞核和细胞膜。实时荧光定量PCR检测分析表明,干旱、高温、盐胁迫和氮胁迫等非生物胁迫均能调控Fa6-SFT基因的表达,推测高羊茅Fa6-SFT可能与非生物逆境胁迫密切相关。Sucrose:fructan 6-fructosyl-transferase(6-SFT) is a key protease of fructan synthesis in Poaceae plants, which plays an important role in plant resistance to drought, heat, salt and nitrogen stresses. In this study, to explore the molecular mechanism of 6-SFT gene under adversity and stress conditions in tall fescue(Festuca arundinacea), a full length cDNA encoding 6-SFT, designated as Fa6-SFT, was cloned from the leaf of tall fescue cultivar ‘Qiancao No.1’ by reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE) methods. Sequence analysis showed that the full-length cDNA of Fa6-SFT gesne was 2 375 bp, containing open reading frame of 1 860 bp in length, which encoding 584 amino acids;the molecular weight of the protein was 68.20 kDa;and the isoelectric point(pI) was 5.61, belonging to the acidic protein. The deduced amino acids contain GH32 family conservative domains. Multiple sequence alignment and phylogenetic analysis showed that the Fa6-SFT protein had high homology with 6-SFT proteins from Phleum pratense, Lolium perenne and L. temulentum. Subcellular localization analysis showed that Fa6-SFT protein was mainly localized in the cell nucleus and cell membrane. The expression of Fa6-SFT gene in the leaf of tall fescue was detected by qRT-PCR. The results indicated that the expression level of Fa6-SFT gene was regulated by drought, heat, salt, and nitrogen stresses. It was speculated that Fa6-SFT gene was associated with abiotic stresses in tall fescue.

关 键 词：Fa6-SFT 基因克隆 生物信息学分析 亚细胞定位 表达分析 

分 类 号：S688.4[农业科学—观赏园艺]