黄肿树JcFATA基因组织表达特性及其启动子克隆与表达分析
发布时间：2018-07-31 20:21
【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(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。
[Abstract]:A (fatty acyl carrier thioesterase (A (fatty acyl carrier thioesterase) plays an important role in fatty acid synthesis and metabolism. Up to now, the expression patterns of (JcFATA) and its promoter of (Jatropha curcas) lipoacyl-ACP thioesterase A gene have not been reported. In order to study the expression pattern of JcFATA gene, the temporal and spatial expression specificity of JcFATA gene was detected by semi-quantitative RT-PCR and real-time fluorescence quantification (qRT-PCR). The results showed that JcFATA gene was expressed in many tissues, 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. The fusion expression vector (p1300G-PJcFATA-GUS) of 尾 -glucuronidase Gus (尾 -glucuronidase Gus) driven by JcFATA gene promoter was successfully constructed, and the recombinant plasmid was transformed into wild type Arabidopsis thaliana (Arabidopsis thaliana), through hygromycin screening by means of Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated transformation. The results of PCR and GUS histochemical analysis showed that the promoter fragment of JcFATA gene had been successfully integrated into Arabidopsis thaliana genome (JGD accession number: Jcr4S00539). The results of GUS histochemical staining of Arabidopsis thaliana plants at different developmental stages showed that the activity of GUS had obvious space-time specificity and had strong GUS activity in roots, flowers and leaves. The results provide a theoretical basis for further study of JcFATA gene and its promoter function.
【作者单位】： 广东海洋大学农学院;嘉应学院医学院;
【基金】：国家自然科学基金项目(No.31271759) 湛江市科技攻关项目(No.2016B101)
【分类号】：Q943.2;S794.9
[Abstract]:A (fatty acyl carrier thioesterase (A (fatty acyl carrier thioesterase) plays an important role in fatty acid synthesis and metabolism. Up to now, the expression patterns of (JcFATA) and its promoter of (Jatropha curcas) lipoacyl-ACP thioesterase A gene have not been reported. In order to study the expression pattern of JcFATA gene, the temporal and spatial expression specificity of JcFATA gene was detected by semi-quantitative RT-PCR and real-time fluorescence quantification (qRT-PCR). The results showed that JcFATA gene was expressed in many tissues, 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. The fusion expression vector (p1300G-PJcFATA-GUS) of 尾 -glucuronidase Gus (尾 -glucuronidase Gus) driven by JcFATA gene promoter was successfully constructed, and the recombinant plasmid was transformed into wild type Arabidopsis thaliana (Arabidopsis thaliana), through hygromycin screening by means of Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated transformation. The results of PCR and GUS histochemical analysis showed that the promoter fragment of JcFATA gene had been successfully integrated into Arabidopsis thaliana genome (JGD accession number: Jcr4S00539). The results of GUS histochemical staining of Arabidopsis thaliana plants at different developmental stages showed that the activity of GUS had obvious space-time specificity and had strong GUS activity in roots, flowers and leaves. The results provide a theoretical basis for further study of JcFATA gene and its promoter function.
【作者单位】： 广东海洋大学农学院;嘉应学院医学院;
【基金】：国家自然科学基金项目(No.31271759) 湛江市科技攻关项目(No.2016B101)
【分类号】：Q943.2;S794.9
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