橡胶树胶乳HbHSP90.6基因克隆、表达与亚细胞定位分析  

Analysis of HbHSP90.6 Gene Cloning,Expression and Subcellular Localization in Rubber Tree Latex

作　　者：刘明洋 杨洪[1] 代龙军[1] 王立丰[1] 郭冰冰 LIU Mingyang;YANG Hong;DAI Longjun;WANG Lifeng;GUO Bingbing(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/Danzhou Investigation&Experiment Station of Tropical Crops,Ministry of Agriculture and Rural Affairs/Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China)

机构地区：[1]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/农业农村部儋州热带作物科学观测实验站/中国热带农业科学院橡胶研究所,海南海口571101

出　　处：《热带作物学报》2023年第10期1935-1943,共9页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金青年基金项目(No.322QN408)。

摘　　要：HSP90是维持细胞内稳态的关键成分,其通过调节其底物的成熟、稳定性、活性和翻转来控制多个细胞过程。橡胶树(Hevea brasiliensis Müll.Arg.)是生产天然橡胶的重要来源,其拥有数千种工业用途,具有较高的社会经济价值。利用PCR技术从橡胶树品种热研73397胶乳中克隆1个HSP90家族成员基因,命名为HbHSP90.6基因(GenBank登录号:OP375588),序列分析表明其编码区(CDS)为2112 bp。理化性质分析表明其相对分子量为80774.85 Da,等电点为5.04,不稳定系数为38.98,总平均亲水性为-0.610,推测HbHSP90.6蛋白是一个稳定的亲水蛋白。保守结构域分析和多序列比对结果显示,HbHSP90.6含有1个PTZ00272的结构域和包含高度保守的MEEVD基序,属于HSP90家族成员。进化分析结果显示,HbHSP90.6与拟南芥AtHSP90.1(NM_124642.4)和水稻OsHsp90-1(LOC_Os04g01740)聚在一起,同源性分别为92.34%和86.44%。组织表达分析显示:HbHSP90.6基因在胶乳中的基因表达量显著高于根、花、枝、茎、叶,HbHSP90.6基因在胶乳中的表达量相对于花达到283倍,推测HbHSP90.6基因可能参与乳管胞内运输和胶乳代谢调控;胶乳中的伤害处理显著上调HbHSP90.6基因的表达,是处理前的19倍,推测HbHSP90.6基因参与机械伤害调控的橡胶树生理与代谢活动反应;HbHSP90.6基因在植物激素乙烯利、茉莉酸处理和吲哚-3-乙酸处理的6 h时表达量均达到最高,分别是处理前的45倍、17倍和6倍;在油菜素内酯处理后的12 h时,HbHSP90.6基因表达量显著上调表达,达到处理前的50倍,推测HbHSP90.6基因参与植物激素介导的转录调控。通过构建35S::HbHSP90.6::GFP融合表达载体进行亚细胞定位,结果显示HbHSP90.6基因定位在细胞质和细胞核中,与亚细胞定位预测结果一致。该研究结果为阐明HbHSP90.6基因在植物激素信号调控的橡胶树逆境胁迫和胶乳代谢分子机制研究奠定坚实基础。HSP90 is a key component in maintaining intracellular homeostasis,and it controls multiple cellular processes by regulating the maturation,stability,activity and turnover of its substrates.Rubber tree(Hevea brasiliensis Müll.Arg.)is an important source for the production of natural rubber,which has thousands of industrial uses and a high socioeco-nomic value.Using PCR technique,an HSP90 family member gene,named HbHSP90.6 gene(GenBank accession number:OP375588),was cloned from rubber tree variety Reyan73397 latex,and the sequence analysis showed that its coding region(CDS)was 2112 bp.Physicochemical analysis showed that its relative molecular weight was 80774.85 Da,isoelectric point was 5.04,instability coefficient was 38.98,and total average hydrophilicity was-0.610,presumably HbHSP90.6 protein is a stable hydrophilic protein.The results of conserved structural domain analysis and multiple sequence alignment showed that HbHSP90.6 contained a structural domain of PTZ00272 and a highly conserved MEEVD motif,which is a member of the HSP90 family.Phylogenetic analysis showed that HbHSP90.6 clustered with Arabidopsis AtHSP90.1(NM_124642.4)and rice OsHsp90-1(LOC_Os04g01740)with 92.34%and 86.44%homology,respectively.Tissue expression analysis showed that the gene expression of HbHSP90.6 gene in latex was significantly higher than that in root,flower,branche,stem and leaf,and the expression of HbHSP90.6 gene in latex reached 283-fold relative to flowers,presumably the HbHSP90.6 gene may be involved in intracellular transport of latex tubes and regu-lation of latex metabolism.Mechanical wounding treatment in latex significantly upregulated the expression of HbHSP90.6 gene by 19 fold compared to the pretreatment pair,and it is hypothesized that HbHSP90.6 gene is involved in mechanical wounding regulated physiological and metabolic activity responses in rubber trees.The expression of HbHSP90.6 gene reached the highest level at 6 h of treatment with the phytohormones ethephon,jasmonic acid and 3-Indoleacetic acid,which were

关 键 词：橡胶树 HbHSP90.6 油菜素内酯 表达分析 亚细胞定位 

分 类 号：S794.1[农业科学—林木遗传育种]