核桃JrHSP20-1基因鉴定及温度胁迫响应功能分析  

On Clone and Expression Analysis of JrHSP20-1 Gene in Juglans Regia

作　　者：孙宇栋[1] 杨桂燕[1] 贾彩霞[1] 胡刁 翟梅枝[1] 

机构地区：[1]西北农林科技大学林学院/核桃研究中心,陕西杨凌712100

出　　处：《西南师范大学学报（自然科学版）》2016年第6期90-95,共6页Journal of Southwest China Normal University(Natural Science Edition)

基　　金：国家公益性林业行业专项(201004027)

摘　　要：探讨核桃(Juglans regia)热激蛋白响应温度胁迫的分子调控机理,为核桃防寒工作等提供理论指导.通过对核桃‘香玲’转录组分析获得1个热激蛋白家族基因HSP20(命名为JrHSP20-1)的全长cDNA序列.采用实时荧光定量PCR(qRT-PCR)技术分析在40,44,48,16,10,6℃胁迫下JrHSP20-1在核桃根、茎、叶中的表达.并将JrHSP20-1构建酵母表达载体PYES2-JrHSP20-1进行温度胁迫功能分析.结果显示,JrHSP20-1基因全长891bp,编码的蛋白分子量为33.57KD,含296个氨基酸,理论等电点为5.07.表达分析显示,JrHSP20-1在不同组织均有不同程度的表达;根中,44℃时JrHSP20-1被诱导为对照的2.3倍,48℃时达对照的74.5倍,10℃时被诱导为对照的12.0倍;茎中,48℃时上调最大为对照的300.2倍;叶中,在48℃和16℃胁迫下被诱导较高,分别为对照的82.1,53.8倍.酵母温度胁迫实验发现,在53℃和-20℃胁迫下,重组酵母INVSC1(pYES2-JrHSP20-1)的生命活力及恢复活性明显优于对照酵母INVSC1(pYES2).得出核桃JrHSP20-1基因的表达具有一定的组织特性,响应于温度胁迫诱导,且JrHSP20-1基因能有效提高转基因酵母对高温和低温的耐受力,表明JrHSP20-1基因是核桃抗寒耐高温的有效候选基因,为进一步研究JrHSP20-1的抗寒功能打下基础.Heat shock proteins（HSP）are important abiotic stresses response proteins in plants.This study is to understand the temperature response molecular mechanism and supply powerful theoretical guidance for Juglans regia cold resistance.The full length cDNA of HSP20（named JrHSP20-1）was isolated and determined fromJuglans regiatranscriptome cDNA library.The expressions of JrHSP20-1in root,stem and leaves exposed to 40,44,48,16,10,6℃ have been analyzed by quantitative real time PCR（qRTPCR）.The recombinant plasmid PYES2-JrHSP20-1has beenconstructed by inserting the JrHSP20-1gene into the yeast expression vector pYES2 to analyze the temperature stresses regulation function.The results showed that the ORF of JrHSP20-1cDNA was 891 bp,encoding a 33.57 KD protein with 296 amino acid residues and the heoretical pI was 5.07.JrHSP20-1is expressed in 3tissues,and in roots,JrHSP20-1is induced to 2.3-fold under 44℃and 74.5-fold expose to 48℃of control,while when treated with 10℃,it is induced to 12.0-fold of control.In stems,JrHSP20-1is up-regulated to a highest level300.2-fold of control respectively.In leaves,JrHSP20-1is induced in a much higher level exposed to 48℃as well as 16℃ with 82.1-fold and 53.8-fold of control.Under 53℃ and-20℃,INVSC1（pYES2-JrHSP20-1）shows better tolerance than control INVSC1（pYES2）.It is concluded that the expression of JrHSP20-1displays tissue specificity and responses to hot and cold stress,JrHSP20-1effectively improves hot and cold tolerance in transgenic yeast,indicating the JrHSP20-1gene is an effective candidate gene for J.regia heat and cold tolerance,and benefits for further understanding the cold tolerance of JrHSP20-1.

关 键 词：核桃 热激蛋白 组织表达 低温胁迫 酵母表达 

分 类 号：Q943.2[生物学—植物学]