橡胶树硫氧还蛋白基因HbTRXo2克隆、表达及功能分析  

Cloning,Expression and Functional Analysis of the Thioredoxin Gene HbTRXo2 in Hevea brasiliensis

作　　者：杨娜 吴双 逯锐琳 何其光 胡义钰[1] 朱春梅 王真辉[1] 刘辉[1] YANG Na;WU Shuang;LU Ruilin;HE Qiguang;HU Yiyu;ZHU Chunmei;WANG Zhenhui;LIU Hui(Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture and Rural Affairs/State Key Laboratory Incubation Base for Cultivation&Physiology of Tropical Crops,Haikou,Hainan 571101,China;College of Tropical Crops,Yunnan Agricultural University,Pu’er,Yunnan 665099,China;School of Tropical Agriculture and Forestry,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]中国热带农业科学院橡胶研究所/农业农村部橡胶树生物学与遗传资源利用重点实验室/省部共建国家重点实验室培育基地-海南省热带作物栽培生理学重点实验室,海南海口571101 [2]云南农业大学热带作物学院,云南普洱665099 [3]海南大学热带农林学院,海南海口570228

出　　处：《热带作物学报》2024年第10期1999-2009,共11页Chinese Journal of Tropical Crops

基　　金：海南省重点研发计划项目(No.ZDYF2024XDNY232);国家自然科学基金面上项目(No.32371922);中央级公益性科研院所基本科研业务费专项(No.1630022023008)。

摘　　要：硫氧还蛋白(thioredoxin,TRX)作为氧化还原调节蛋白在植物抵御非生物逆境胁迫中发挥重要作用。本研究采用RT-PCR从橡胶树(Heveabrasiliensis)中克隆了硫氧还蛋白基因HbTRXo2,该基因编码区长594bp,编码197个氨基酸。预测HbTRXo2蛋白的分子量为21.90 kDa,理论等电点为7.59。蛋白保守结构域和系统进化分析结果显示,HbTRXo2含有TRX保守结构域,与其他植物o型硫氧还蛋白聚在一起,表明该蛋白属于o型硫氧还蛋白。实时荧光定量PCR分析表明,HbTRXo2基因在橡胶树根、树皮、胶乳、成熟叶、衰老叶、新梢、雌花和雄花组织中均表达,其中在胶乳中的表达量显著高于其他组织。同健康橡胶树相比,割面干涸橡胶树树皮和胶乳中HbTRXo2的表达量显著降低。在低温、聚乙二醇(PEG)诱导的干旱以及过氧化氢(H_(2)O_(2))和甲基紫精(MV)诱导的氧化胁迫处理下,HbTRXo2基因的表达显著上调,表明该基因参与了橡胶树对非生物胁迫的应答。为了研究HbTRXo2在抗逆中的功能,本研究构建其酵母表达载体,并转入酿酒酵母INVSc1菌株中,获得重组酵母INVSc1(pYES2-HbTRXo2)。比较重组酵母INVSc1(pYES2-HbTRXo2)和转空载体对照酵母INVSc1(pYES2)在H_(2)O_(2)、PEG和低温胁迫处理后的存活差异。结果显示,重组酵母INVSc1(pYES2-HbTRXo2)在PEG和H_(2)O_(2)处理后的存活率明显高于对照酵母,而在低温胁迫处理后的存活率明显低于对照酵母,表明转HbTRXo2基因提高了重组酵母对干旱和氧化胁迫的抗性,但降低了对低温胁迫的抗性。以上研究结果证明,HbTRXo2在橡胶树产排胶和抗逆过程中起着重要作用。本研究为进一步解析HbTRXo2在橡胶树中的生物学功能提供重要的参考依据。Thioredoxin(TRX),a redox regulatory protein,plays an important role in plant resistance to abiotic stress.In this study,HbTRXo2,a thioredoxin gene,was cloned from rubber tree(Hevea brasiliensis)using RT-PCR.HbTRXo2 contained a coding region of 594 bp encoding a protein of 197 amino acids.The predicted molecular weight and isoelectric point of HbTRXo2 was 21.90 kDa and 7.59,respectively.The conserved domain and phylogenetic analysis showed that HbTRXo2 had a conserved TRX domain and was clustered with other plant o-type thioredoxin,suggesting that HbTRXo2 belonged to o-type thioredoxin.Quantitative real-time PCR indicated that HbTRXo2 gene was expressed in root,bark,latex,mature leaf,senescent leaf,new shoot,female flower and male flower tissues of rubber tree,with significantly higher expression level in latex than in other tissues.Compared with the healthy rubber trees,the expression of HbTRXo2 in the bark and latex of tapping panel dryness trees was significantly reduced.Under cold,polyethylene glycol(PEG)-induced drought,and oxidative stress induced by hydrogen peroxide(H_(2)O_(2))and methyl violet(MV),the expression of HbTRXo2 gene was significantly up-regulated,indicating the involvement of HbTRXo2 in response to abiotic stress in rubber tree.To explore the function of HbTRXo2 in stress resistance,its yeast expression vector was constructed and transferred into Saccharomyces cerevisiae INVSC1 to obtain the recombinant yeast INVSC1(pYES2-HbTRXo2).The survival differences between the recombinant yeast INVSC1(pYES2-HbTRXo2)and the control yeast INVSC1(pYES2)transformed with pYES2 empty vector after H_(2)O_(2),PEG,and low temperature stress treatments were compared,and the results showed that the survival rate of INVSc1(pYES2-HbTRXo2)was significantly higher than that of INVSC1(pYES2)after PEG and H_(2)O_(2) treatments,while the survival rate of INVSc1(pYES2-HbTRXo2)was significantly lower than that of INVSC1(pYES2)after low temperature stress treatment,indicating that the recombinant yeast transformed with HbTR

关 键 词：硫氧还蛋白 橡胶树 基因表达 酵母表达 抗氧化性 非生物逆境胁迫 

分 类 号：S794.1[农业科学—林木遗传育种]