论文部分内容阅读
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(PJcFATA)插入到双元表达载体pCAMBIA1300G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。
The fatty acyl acyl carrier thioesterase (FATA) plays an important regulatory role in the synthesis and metabolism of fatty acids. At present, no reports have been reported on the expression pattern of Jatropha curcas fatty acyl-ACP thioesterase A gene (JcFATA) and its promoter. In order to study the expression pattern of JcFATA gene, this study detected the temporal and spatial expression specificity of JcFATA gene by semi-quantitative RT-PCR and real-time fluorescence quantitative (qRT-PCR). The results showed that JcFATA gene was expressed in many tissues of the tree, especially in roots, flowers and leaves. In order to analyze the tissue expression pattern of JcFATA gene promoter, the cloned JcFATA gene promoter fragment (PJcFATA) was inserted into the binary expression vector pCAMBIA1300G and the JcFATA gene promoter-driven β-glucuronidase β-glucuronidase (GUS) gene fusion expression vector (p1300G-PJcFATA-GUS). The recombinant plasmid was transformed into wild-type Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation, Finally, the resistant plants were tested by PCR and GUS histochemical analysis. PCR results showed that the JcFATA gene promoter fragment has been successfully integrated into Arabidopsis genome (JGD accession number: Jcr4S00539). GUS histochemical staining of Arabidopsis plants at different developmental stages showed that GUS activity had obvious spatio-temporal specificity, deep staining in roots, flowers and leaves, and strong GUS activity. The results of this study provide a theoretical basis for further study of JcFATA gene and its promoter function.