橡胶树乳管表达HbHMGR1基因双元表达载体构建与鉴定  

Construction and identification of binary vector containing HbHMGR1 gene from laticifer of Hevea brasiliensis

作　　者：贺永国 李哲[2] 黄绵佳[1] 曾宪海[2] 林位夫[2] 刘洁琼[2] 张春红[2] 马晓晓[3] 

机构地区：[1]海南大学园艺园林学院,海口570228 [2]中国热带农业科学院橡胶研究所/农业部橡胶树生物学与遗传资源利用重点实验室,海南儋州571737 [3]海南大学农学院,海口570228

出　　处：《南方农业学报》2016年第2期163-168,共6页Journal of Southern Agriculture

基　　金：中国热带农业科学院基本科研业务费专项项目(1630022013029)

摘　　要：【目的】克隆橡胶树乳管特异性强启动子HEV2.1(PHEV2.1),构建天然橡胶生物合成途径关键限速酶基因(Hb HMGR1)的乳管特异性植物表达载体,为橡胶树遗传转化奠定基础。【方法】根据已报道的HEV2.1序列(Gen Bank登录号AY247789.1)设计1对特异引物,采用PCR从橡胶树热研7-33-97基因组DNA中克隆PHEV2.1,与Hb HMGR1基因融合构建橡胶树乳管特异性植物表达载体,并以PCR和限制性内切酶酶切进行鉴定。【结果】用特异引物可从热研7-33-97基因组DNA中扩增获得1852 bp的PHEV2.1片段,其与AY247789.1启动子序列的同源性为98.6%。将PHEV2.1与Hb HMGR1基因融合构建乳管特异性植物表达载体p CAMBIA2301-PHEV2.1-Hb HMGR1,经PCR和限制性内切酶酶切鉴定证明植物表达载体构建成功。【结论】将PHEV2.1和Hb HMGR1基因先构建到p CAMBIA3301载体上再克隆至p CAMBIA2301载体上,能有效解决直接克隆至p CAMBIA2301载体上出现Pst I突变、阻碍载体构建的难题,成功构建获得乳管特异性植物表达载体p CAMBIA2301-PHEV2.1-Hb HMGR1。【Objective】The present experiment was conducted to clone specific strong promoter HEV2.1（PHEV2.1） of Hevea brasiliensis laticifer and construct laticifer＇s specific plant expression vector containing key rate-limiting enzyme gene Hb HMGR1 in natural rubber biosynthesis, in order to lay the foundation for further genetic transformation of H.brasiliensis. 【Method】 According to reported sequence of HEV2.1（Gen Bank Accession NO. AY247789.1）, a pair of specific primers was designed. Then the promoter（PHEV2.1） of HEV2.1 gene was cloned from genomic DNA of H. brasiliensis Reyan 7-33-97 by PCR method, and which was fused with Hb HMGR1 gene to construct plant expression vector p CAMBIA2301-PHEV2.1- Hb HMGR1. Further, the constructed plant expression vector was identified by PCR method and restriction enzyme digestion. 【Result 】The results showed that, the specific promoter PHEV2.1 fragment was cloned from genomic DNA of Reyan 7-33-97, which was 1852 bp in length and shared homology of 98.6% with HEV2.1 promoter sequence of AY247789.1. Then the plant expression vector p CAMBIA2301-PHEV2.1-Hb HMGR1 was constructed by fusing PHEV2.1 and Hb HMGR1, and the PCR, and restriction enzymes digestion results showed that the plant expression vector was constructed successfully. 【Conclusion】PHEV2.1 and Hb HMGR1 were linked with p CAMBIA3301, then cloned into p CAMBIA2301, so as to avoid Pst I mutation in linkage with p CAMBIA2301 and solve difficult problem in vector construction. Thus, the laticifer specific plant expression vector p CAMBIA2301-PHEV2.1-Hb HMGR1 was constructed successfully.

关 键 词：橡胶树 乳管特异性启动子PHEV2.1 HbHMGR1基因 植物表达载体 构建 鉴定 

分 类 号：S794.1[农业科学—林木遗传育种]