油棕脂肪酸脱饱和酶基因ω3启动子区的克隆及其表达组织特异性分析

Cloning and Expression Tissue-specific Analiysis of Fatty Acid Desaturase Gene ω3 Promoter from Oil Palm

本研究是以油棕叶片基因组DNA为模板,通过数据库预测的油棕脱饱和酶基因ω3的启动子序列设计特异性引物,利用PCR技术扩增及TA克隆的方法得到了长度为1 144 bp油棕脱饱和酶基因ω3启动子序列。通过Plant CARE软件分析该序列的顺式作用元件,发现该启动子不仅含有转录必备的CAAT-box和TATA box,还有大量相关的光反应元件和激素响应元件。通过构建含gus的植物表达载体,农杆菌介导转化转基因拟南芥,对转基因拟南芥进行活性表达分析。结果表明:克隆获得的启动子序列和下游gus基因已经稳定的整合到获得的转基因拟南芥基因组当中;同时,gus基因在拟南芥不同组织中体现出明显的组织特异性,其中,茎干处表达量最高,在叶片的叶脉处表达次之。说明ω3启动子具有活性,可以启动目的基因的转录,本研究为进一步研究启动子的功能及基因表达调控奠定了基础。

In the study,oil palm genomic DNA was used as template and specific primers were designed based on gene sequence of fatty acid desaturase gene ω3 promoter in oil palm predicted by genome database.The ω3 gene potential promoter region was amplified by PCR and TA cloning.Analyses of the promoter revealed that the length of the promoter sequence is 1 144 bp.The cis-acting elements of the sequence were analyzed by Plant CARE software.It was found that the promoter not only harbored CAAT box and TATA box,but also possessed multiple responsive elements and hormone responsive elements.The activity expression of transgenic arabidopsis was analyzed by structuring plant expression vector which contains Gus and being transformed by Agrobacterium mediated transformation.The result showed that the colned promoter sequences and downstream of the integration of gus gene has been stable to obtain transgenic arabidopsis genome.In the meanwhile,gene Gus showed obvious tissue specificity in different tissues.The results showed that gus gene is highly expressed in Arabidopsis stem and secondly expressed in leaf veins.Consequently,the promoter activity of ω3 can be indicated the transcription of the target gene.This study might provide a base for further investigating the function and transcriptional regulation of promoter.