橡胶树乳管细胞GPPS基因的克隆与表达分析  

Cloning and expression analysis of GPPS genes from the laticifer cells of Hevea brasiliensis

作　　者：邓小敏[1] 于洁 陈莹 晁金泉[1] 张世鑫[1] 田维敏[1] DENG Xiao-min;YU Jie;CHEN Ying;CHAO Jin-quan;ZHANG Shi-xin;TIAN Wei-min(1Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Biology,Ministry of Agriculture and Rural Affairs/Hainan Key Laboratory for Cultivation and Physiology of Tropical Crops,Danzhou,Hainan 571737,China;Institute of Tropical Agriculture and Forestry,Hainan University,Danzhou,Hainan 571737,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/海南省热带作物栽培生理学重点实验室,海南儋州571737 [2]海南大学热带农林学院,海南儋州571737

出　　处：《南方农业学报》2018年第11期2117-2128,共12页Journal of Southern Agriculture

基　　金：国家现代农业产业技术体系(天然橡胶)建设专项资金项目(CARS-34-GW1);中国热带农业科学院橡胶研究所基本科研业务费专项项目(1630022015010;1630022016003)

摘　　要：【目的】克隆橡胶树乳管细胞短链异戊烯基合酶(GPPS)基因,分析其表达特性和编码蛋白的相互作用,为解析该类基因在天然橡胶和萜类合成中的作用提供理论参考。【方法】采用逆转录PCR(RT-PCR)和cDNA末端快速克隆(RACE)从橡胶树乳管细胞胶乳中克隆GPPS基因(HbGPPS1和HbGPPS2),利用生物信息学分析其编码蛋白的特性;采用实时荧光定量PCR(q RT-PCR)检测HbGPPS1和HbGPPS2在树皮和胶乳中的组织表达特异性、在不同橡胶树品系中的表达模式及其对割胶和外源茉莉酸甲酯(MeJA)处理的响应模式,并在原核细胞中进行表达分析;通过酵母双杂交技术检测HbGPPS1和HbGPPS2蛋白的相互作用关系。【结果】从橡胶树乳管细胞乳胶中克隆获得HbGPPS1和HbGPPS2基因,其中HbGPPS1基因编码框长度为1248 bp,编码415个氨基酸,蛋白分子量为45.9 kD,理论等电点为6.77;HbGPPS2基因编码框长度为1239 bp,编码412个氨基酸,蛋白分子量为45.6 kD,理论等电点为6.77。二者的核苷酸序列相似性为89%,且编码蛋白均含有2个典型的异戊烯基转移酶活性结构域DDXXD(D),定位于叶绿体和线粒体。HbGPPS1蛋白自身及其与HbGPPS2蛋白均能形成较弱相互作用的二聚体。qRT-PCR检测结果显示,HbGPPS1和HbGPPS2基因在胶乳中的表达量高于树皮,但均以HbGPPS2基因表达量较高,表明二者表达存在组织特异性。经MeJA处理后橡胶树胶乳中HbGPPS1和HbGPPS2基因表达量较对照高,即外源MeJA可促进内源茉莉酸的合成,激活乳管细胞内的茉莉酸信号从而上调HbGPPS1和HbGPPS2基因表达。在5个橡胶树栽培品系中,HbGPPS2基因的表达量均明显高于HbGPPS1基因,且与PR107、热研7-20-59、RRIM600和热研7-33-97干胶含量趋势一致,但二者仅在热研8-79和热研7-33-97的表达模式存在差异。HbGPPS2基因能在大肠杆菌中成功表达。【结论】HbGPPS2基因可能是乳管细胞中参与天然橡胶和萜类合成的主效基因,�【Objective】The short-chain isopentenyl synthase(GPPS)genes from the laticifer cells of Hevea brasiliensis were cloned and interaction between their expression patterns and encoding proteins were further analyzed in this study for dissecting the function of the GPPS genes in the biosynthesis of natural rubber and terpenoids.【Method】Reverse transcription-PCR(RT-PCR)and rapid amplification of cDNA ends(RACE)methods were performed to clone the GPPS genes from the laticifer cells of H.brasiliensis.The character of GPPS genes encoding proteins were analyzed by bioinformatics.Moreover,real-time fluorescence quantitative PCR(qRT-PCR)was used to assess tissue-specific expression of GPPS genes from H.brasiliensis in the bark and latex and expression pattern of GPPS genes in different clones of H.brasiliensis.qRT-PCR was also used to detect response models of GPPS genes to tapping and exogenous methyl jasmonate(MeJA)treatment.And expression analysis of GPPS-encoding genes was done in prokaryotic cells.The protein interactions of HbGPPS1 and HbGPPS2 proteins were detected by using yeast two-hybrid technique.【Result】Genes HbGPPS1 and HbGPPS2 were successfully cloned from the latex of laticifers cells in H.brasiliensis.HbGPPS1 gene harboured a coding frame of 1248 bp in length that encoded 415 amino acids with the protein molecular weight of 45.9 kD and the theoretical isoelectric point(pI)of 6.77.While the gene HbGPPS2 harboured a coding frame of 1239 bp in length that encoded 412 amino acids with the protein molecular weight of 45.6 kD and the pI of 6.77.The similarity of their nucleotide sequence was up to 89%.The encoding proteins harbored two active prenyl transferase domain DDXXD(D)which were located in chloroplast and mitochondria.Protein interaction assay demonstrated that HbGPPS1 protein harbored relatively weak interaction with itself or with HbGPPS2 protein.Detection results of qRT-PCR revealed that both HbGPPS1 and Hb-GPPS2 had higher expression levels in latex than that in the bark,and the HbGPPS2 gene

关 键 词：橡胶树 乳管细胞 短链异戊烯基合酶(GPPS) 基因克隆 表达水平 蛋白相互作用 

分 类 号：S794.1[农业科学—林木遗传育种]