巴西橡胶HbWRKY55启动子的克隆及功能初步分析  

Cloning and Preliminary Function Analysis of the Promoter of HbWRKY55 from Hevea brasiliensis

作　　者：曲龙 张建平[2] 李辉亮[2] 郭冬[2] 罗丽娟 彭世清[2] Qu Long;Zhang Jianping;Li Huiliang;Guo Dong;Luo Lijuan;Peng Shiqing(College of Agronomy, Hainan University, Haikou, 570228;Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agri- culture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101)

机构地区：[1]海南大学热带农林学院,海口570228 [2]中国热带农业科学院热带生物技术研究所,农业部热带作物生物学与遗传资源利用重点实验室,海口571101

出　　处：《分子植物育种》2018年第9期2827-2833,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31670611); 中国热带农业科学院基本科研业务费专项资金(1630052016003)共同资助

摘　　要：为深入研究巴西橡胶树HbWRKY55调控天然橡胶生物合成机制,本研究根据已克隆的巴西橡胶树HbWRKY55的序列和橡胶树基因组序列信息设计引物,通过PCR技术获得了HbWRKY55起始密码子ATG上游1 587 bp的序列,利用Plant CARE数据库对该序列的调控元件进行分析。分析结果表明该启动子序列除具有多个典型的真核生物启动子基本元件如增强子元件CAAT-box、启动子核心序列TATA-box等外,还存在赤霉素反应元件GARE-motif、P-box以及水杨酸响应元件TCA-element等参与激素调控的应答元件,同时光应答元件BoxⅠ、Gap-box、Ⅰ-box、L-box、GAG-motif和GT1-motif,胚乳表达的调控元件GCN4-motif、Skn-1_motif和厌氧诱导元件ARE等顺式作用元件也包含其中。构建了该序列的4个5'端缺失片段和荧光素酶基因融合的表达载体,瞬时表达结果表明赤霉素(GA)、水杨酸(SA)可以抑制HbWRKY55启动子的活性,脱落酸(ABA)、茉莉酸甲脂(MeJA)可以加强HbWRKY55启动子的活性。在HbWRKY55启动子的-641^-455和-237^+1可能存在抑制ABA的元件;HbWRKY55启动子的-454^-238和-66^+1存在JA应答增强元件。本研究为深入了解HbWRKY55的调控机理提供帮助。In order to further study the mechanism of HbWRKY55 in regulating natural rubber biosynthesis in Hevea brasiliensis, primers were designed based on the cloned sequence of HbWRKY55 in Hevea brasiliensis and the genomic sequence information of Hevea brasiliensis. A 1 587 bp sequence in the upstream of the initiation codon ATG of HbWRKY55 was cloned by using PCR techniques, and its regulatory elements were analyzed by Plant CARE website database. The results showed that this promoter sequence not only contained several typical eukaryote promoter elements, such as enhancer element CAAT-box, and promoter core sequence TATA-box, but also contained several response elements involved in hormone regulation, including GARE-motif, P-box and TCA-element. At the same time, some cis-acting elements, such as light responsive element Box I, Gap-box, I-box,L-box, GAG-motif, GT1-motif, regulatory element of the endosperm expression GCN4-motif, Skn-1_ motif and anaerobic inducer ARE, were also found in the promoter region of HbWRKY55. Four plant expression vectorswith truncated promoter fragments and a luciferase reporter gene were constructed, and the transient expression results indicated that gibberellin（GA） and salicylic acid（SA） could inhibit the activity of HbWRKY55 promoter,while abscisic acid（ABA） and methyl jasmonate（MeJA） could enhance the activity of HbWRKY55 promoter.Elements that could inhibit ABA might exist in-641 to-455 and-237 to ＋1 of HbWRKY55 promoter. There was a JA response enhancer in-454 to-238 and-66 to ＋1 of HbWRKY55 promoter. This research could be helpful for further understanding of the regulation mechanism of HbWRKY55.

关 键 词：橡胶树 HbWRKY55 5'缺失体片段 顺式调控元件 

分 类 号：S794.1[农业科学—林木遗传育种]