黄肿树JcFATA基因组织表达特性及其启动子克隆与表达分析

摘要

植物脂酰-酰基载体蛋白硫酯酶A(fatty acyl acyl carrier thioesterase,FATA)在脂肪酸合成代谢过程中具有重要的调控作用.目前有关黄肿树(Jatropha curcas)脂酰-ACP硫酯酶A基因(JcFATA)和其启动子的表达模式均还未见报道.为了研究JcFATA基因的表达模式,本研究利用半定量RT-PCR和实时荧光定量(qRT-PCR)检测了JcFATA基因的时空表达特异性.结果表明,JcFATA基因在黄肿树的多个组织部位都有表达,尤其是在根、花和叶中表达量较高.为了分析JcFATA基因启动子的组织表达模式,本研究将克隆的JcFATA基因启动子片段(PJcFA TA)插入到双元表达载体pCAMBIA 1300G中,成功构建了JcFATA基因启动子驱动的β-葡糖醛酸酶([β-glucuronidase,GUS)基因融合表达载体(p1300G-PJcFATA-GUS);通过农杆菌(Agrobacterium tumefaciens)介导的转化方法将重组质粒转入野生型拟南芥(Arabidopsis thaliana),经过潮霉素筛选,最后对抗性苗进行PCR检测和GUS组织化学分析.PCR检测结果表明,JcFATA基因启动子片段已经成功整合到拟南芥基因组中(JGD登录号:Jcr4S00539).对不同发育时期的拟南芥植株进行GUS组织化学染色分析,结果显示GUS活性具有明显的时空特异性,在根、花和叶中的染色较深,有很强的GUS活性.本研究结果为进一步研究JcFATA基因及其启动子功能提供了理论依据.%Fatty acyl acyl carrier thioesterase A (FATA) plays an important role in the process of fatty acid synthesis metabolism.The expression pattern of FATA of Jatropha curcas (JcFATA) and its promoter all has not been reported.To study the expression pattern of JcFATA gene,semi-quantitative RT-PCR and qRT-PCR were applied to detect the expression specificity of JcFATA gene.The results showed that JcFATA gene could express in multiple tissues of J.curcas,especially highly expressed in roots,flowers and leaves.In addition,in order to analyze the expression pattern of JcFATA gene promoter (PJcFATA),JcFATA gene promoter was cloned firstly and inserted into a binary expression vector pCAMBIA1300G,and then the fusion expression vector of [β-glucuronidase (GUS) gene drived by JcFATA gene promoter (pl300G-PJcFATA-GUS) was constructed successfully.The fusion expression vector was introduced into Arabidopsis thaliana wild type by Agrobacterium-mediated method.After effective selection for hygromycin B resistance,the resistant plants were detected by PCR and GUS histochemical analysis.PCR detection results showed that JcFATA gene promoter had been transferred into the genome of A.thaliana (JGD No.Jcr4S00539).Results of GUS histochemical staining of tissues in different development periods of A.thaliana revealed that GUS activity displayed obviously tissue specificity,with the deeper staining in roots,flowers and leaves.All above results paved the way to further function study ofJcFATA gene and JcFATA gene promoter.