樟叶越桔热激蛋白基因对温度胁迫的响应表达  

Expression of heat shock protein genes of Vaccinium dunalianum in response to temperature stress

作　　者：李雪[1] 邵亚林 陈海涛[1] 赵平[2] 丁勇[1] LI Xue;SHAO Yalin;CHEN Haitao;ZHAO Ping;DING Yong(Key Laboratory of Forest Biotechnology in Yunnan,Southwest Forestry University,Kunming 650224,Yunnan,China;Key Laboratory of State Forestry and Grassland Administration on Highly-efficient Utilization of Forestry Biomass Resources in Southwest China,Southwest Forestry University,Kunming 650224,Yunnan,China)

机构地区：[1]西南林业大学云南省高校林木生物技术重点实验室,云南昆明650224 [2]西南林业大学西南地区林业生物质资源高效利用国家林业与草原局重点实验室,云南昆明650224

出　　处：《中南林业科技大学学报》2022年第2期139-146,168,共9页Journal of Central South University of Forestry & Technology

基　　金：国家自然科学基金项目(31960073,31460076);云南省高校林木生物技术重点实验室开放基金项目(51700201)。

摘　　要：【目的】分析樟叶越桔热激蛋白基因响应不同温度变化的表达模式,为阐明热激蛋白在樟叶越桔响应温度胁迫过程中的生物学功能奠定理论基础。【方法】以樟叶越桔成熟叶片为材料,从建成的转录组数据库中筛选出候选内参基因(VdARP、VdGAPDH和VdEF-1α)和热激蛋白基因(VdHSP90、VdHSP70和VdsHSP)的cDNA序列,利用RT-qPCR技术检测目的基因在不同温度(4、25和35℃)、不同时间(1、5、10和15 d)处理下的表达情况。应用软件BestKeeper、GeNorm、Normfinder和RefFinder综合分析3个候选内参基因的表达稳定性,以表达最稳定的内参基因作为参照,分析3个热激蛋白功能基因响应不同温度的表达模式。【结果】软件BestKeeper、GeNorm、Normfinder和RefFinder综合分析显示,3个候选内参基因表达稳定性为VdARP>VdGAPDH>VdEF-1α,因此以VdARP为内参基因分析热激蛋白基因的表达情况。试验中3个热激蛋白基因在25℃各时间处理组中的表达水平整体趋于稳定。4℃和35℃处理均能诱导樟叶越桔VdHSP90和VdHSP70基因在一定时间内表达水平显著增加,但VdHSP90和VdHSP70基因受4℃诱导表达的水平分别显著高于35℃。VdsHSP基因受35℃高温诱导后显著上调表达,但显著受到4℃低温的抑制而下调表达。【结论】3个热激蛋白基因VdHSP90、VdHSP70和VdsHSP在不同温度(4、25和35℃)处理的樟叶越桔叶片中均有表达,其表达水平显著受到高、低温胁迫的影响。VdHSP90和VdHSP70基因表达在高、低温度胁迫下均显著上调,且对低温胁迫的响应更显著;而VdsHSP基因仅在高温度胁迫下显著上调表达。显示HSP90和HSP70基因表达产物在樟叶越桔抵御零上低温胁迫过程发挥重要功能,而sHSP基因表达产物则主要参与了樟叶越桔对高温胁迫的响应过程。【Objective】In this work,the expression patterns of heat shock protein genes of Vaccinium dunalianum in response to different temperature changes were analyzed,in order to lay a theoretical foundation for the elucidation of their biological functions in response to temperature stress.【Method】The cDNA sequences of three candidate reference genes VdARP,VdGAPDH and VdEF-1αand three heat shock protein genes VdHSP90,VdHSP70 and VdsHSP were obtained from the transcriptome database of V.dunalianum.By taking V.dunalianum mature leaves as the materials,the relative expression levels of these genes under different temperatures(4℃,25℃and 35℃)and different time(1 d,5 d,10 d and 15 d)treatments were measured via real-time fluorescence quantitative PCR(RTqPCR).The expression stability of three candidate internal reference genes was analyzed by BestKeeper,GeNorm,Normfinder and RefFinder softwares,and the most stable transcript was used as the reference to analyze the expression patterns of the three heat shock protein genes of V.dunalianum in response to different temperature treatments.【Result】By using BestKeeper,GeNorm,Normfinder and RefFinder softwares,the comprehensive analysis showed that the stability order of the three candidate reference transcripts was VdARP>VdGAPDH>VdEF-1α.Therefore,VdARP gene was subsequently selected as the internal reference gene to analyze the expression of the heat shock protein genes.The expression levels of the three heat shock protein genes in the treatment groups at 25℃tended to be stable.Temperature treatments at 4℃and 35℃could significantly increase the expression levels of VdHSP90 and VdHSP70 genes in V.dunalianum within a certain period of time.However,the expression levels of VdHSP90 and VdHSP70 genes under 4℃treatment were higher than those at 35℃.The VdsHSP gene expression was significantly up-regulated after being induced by a high temperature of 35℃,but significantly down-regulated by the low temperature of 4℃.【Conclusion】The expression levels of

关 键 词：樟叶越桔 温度胁迫 内参基因 热激蛋白 基因表达 

分 类 号：S759.82[农业科学—森林经理学]