橡胶树HbHMGS1启动子对光照、干旱及热击处理的表达响应  

Expressional Responses of the HbHMGS1 Promoter to Light, Drought and Heat-shock Treatments

作　　者：巩笑笑 闫冰玉 谭玉荣 王鹏 李双江 王艺 周璐瑶 刘进平 GONG Xiaoxiao;YAN Bingyu;TAN Yurong;WANG Peng;LI Shuangjiang;WANG Yi;ZHOU Luyao;LIU Jinping(Tropical Agriculture and Forestry Institute/ Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学热带农林学院/海南省热带生物资源可持续利用重点实验室,海口570228

出　　处：《热带生物学报》2019年第1期52-59,共8页Journal of Tropical Biology

基　　金：国家自然科学基金项目(31560573);海南大学热带农林学院作物学科研究生科研创新课题(ZWCX2018030);海南大学作物学优秀本科生人才培养计划课题(ZWCX2018046)

摘　　要：克隆了橡胶树HbHMGS1基因上游长1 656 bp的启动子序列及其一系列5′缺失片段,并利用PlantCARE启动子预测软件对其进行分析,同时,以GUS基因作为报告基因,构建植物缺失表达载体并通过农杆菌介导的方法转化烟草和拟南芥,并对GUS蛋白的表达活性进行定性及定量检测分析。结果表明:该启动子能够在烟草叶片和T2代转基因拟南芥植株幼苗时期及成熟期不同组织器官中驱动下游GUS基因的表达,且叶片染色呈现明显的蓝色。将包含有HbHMGS1启动子的转基因拟南芥苗进行光周期处理发现,GUS蛋白活性在光照96 h处理时瞬间增大到处理72 h时的3.2倍,黑暗条件下处理72,96 h后叶片GUS活性也显著增加,分别是处理48 h时GUS活性的1.38和2.13倍。通过定量检测HbHMGS1启动子及其各缺失启动子响应热击及干旱处理的GUS蛋白活性发现,-699到起始密码子-1处的699 bp序列可能是主要的热击响应区域;干旱响应区域则主要位于-213到起始密码子-1处的213 bp序列中。结果表明,HbHMGS1启动子在调控HbHMGS1基因响应光照、热击及干旱诱导表达方面起到重要作用。3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) synthase(HMGS) is an important enzyme in the mevalonate(MVA) pathway of isoprenoid biosynthesis, which regulates the rubber biosynthetic pathway in rubber tree(Hevea brasiliensis) in coordination with other genes. However, little information is available about the regulation of HMGS gene expression. The full-length HbHMGS1 promoter was cloned, its promoter sequence analyzed with PlantCARE database, and a series of 5′deletion constructs constructed. The constructs including CaMV35 S-GUS were transformed into tobacco and Arabidopsis thaliana by using the Agrobacterium-mediated method, and characterized quantitatively and ualitatively in transgenic plants with the GUS gene as a reporter gene. The GUS histochemical staining was observed in the leaves of tobacco and different tissues of T2 transgenic Arabidopsis plants at the young and mature developmental stages. When the T2 transgenic Arabidopsis plants containing the HbHMGS1 promoter were exposed to light and darkness, the activity of GUS protein 96 h after light treatment increased quickly to about 3.2 times of that 72 h after light treatment, and the GUS activity of leaves 72 h and 96 h after dark treatment increased to 1.38 and 2.13 times of that 48-h after dark treatment, respectively. Quantitative detection of the GUS protein activity in response to heat shock and drought treatments showed that the 699-bp sequence from-699 to the start codon-1 might be the major heat shock response region, and that the drought response region was mainly located in the 213-bp sequence from-213 to the start codon-1. These results indicated the HbHMGS1promoter may play important roles in regulating of expression of the HbHMGS1 gene in response to light, heat-shock and drought.

关 键 词：橡胶树 羟甲基戊二酰辅酶A合酶1 启动子分析 GUS染色 

分 类 号：Q786[生物学—分子生物学]