檀香NDH脱氢酶基因的克隆、定位与启动子分析

Molecular cloning, location and promoter analysis of NDH dehydrogenase gene from Santalum album

关键词:  檀香, 叶绿体, NDH脱氢酶, 亚细胞定位, 表达调控

Key words:  Santalum album, chloroplast, NDH dehydrogenase, subcellular location, expression regulation

摘要:

为研究檀香NDH脱氢酶基因的功能和调控机制,该文以檀香心材为材料,利用RACE技术克隆SaNDH6基因的全长序列,利用实时荧光定量PCR(RT-qPCR)技术分析其组织和激素处理后的表达模式,在拟南芥原生质体观测其亚细胞定位,利用PlantCARE分析SaNDH6起始密码子ATG上游2 kb的启动子序列,同时运用PlantRegMap预测可能与其结合的转录因子。结果表明:(1)SaNDH6编码303个氨基酸,为疏水蛋白,亚细胞定位于叶绿体。(2)进化树分析表明,檀香SaNDH6与木本植物NDH6进化关系较近。(3)PlantCARE分析发现,SaNDH6启动子中除含有ACE、AE-box、Box 4、G-Box和GT1-motif等大量光响应元件外,同时还有茉莉酸甲酯(MeJA)反应元件CGTCA-motif和TGACG-motif,赤霉素(GA₃)响应元件P-box,以及防御和胁迫响应元件TC-rich repeats等。(4)PlantRegMap分析发现,有76个转录因子可能与SaNDH6启动子结合,其中ERF家族最多,达40个。(5)SaNDH6在檀香的根、心材、叶片和愈伤组织中均有表达,其中在叶片中的表达量较高; 用1×10^-4 mol·L^-1的MeJA和GA₃分别处理檀香愈伤组织后,与处理前(0 h)相比,SaNDH6的表达均在3 h后显著升高。综上结果表明,檀香SaNDH6为核基因编码的蛋白,受光和激素等诱导表达,SaNDH6可能参与檀香逆境胁迫反应的过程。

Abstract:

In order to investigate the function and regulation mechanism of NDH dehydrogenase gene in Santalum album, the technique of RACE was used to amplify the full-length sequence of SaNDH6 with heartwood as material. The technique of quantitative real-time fluorescence PCR(RT-qPCR)was employed to analyze its expression in different tissues and after hormone induction. The subcellular location was determined by Arabidopsis thaliana protoplast transient expression. 2 kb cis-acting element upstream of start codon ATG was analyzed by PlantCARE online service, and the transcription factors which could bind the cis-acting elements was predicted by PlantRegMap software. The results were as follows:(1)SaNDH6 encoded 303 amino acids. It was a hydrophobin and located in chloroplast.(2)The phylogenetic tree analysis indicated that SaNDH6 had a more closely evolutionary relationship with NDH6 from woody plants.(3)Plant care analysis showed that the promoter sequence of SaNDH6 contained a large number of light responsive cis-acting elements such as ACE, AE-box, Box 4, G-Box and GT1-motif. It also contained abscisic acid(ABA)responsive element ABRE, jasmonic acid methyl ester(MeJA)responsive elements CGTCA-motif and TGACG-motif, gibberellin(GA₃)responsive elements P-box, ARE cis-acting regulatory element essential for the anaerobic induction, and TC-rich repeats element involved in defense and stress responsiveness.(4)The results of plantRegMap analysis showed that there were 76 transcription factors that could bind to the SaNDH6 promoter, and among which, ERF transcription factor was the most(up to 40 TFs).(5)SaNDH6 can be expressed in the tissues of roots, heartwoods, calluses and leaves, but had a higher expression level in the tissue of leaves; under 1×10^-4 mol·L^-1 MeJA and GA₃ treatments, the expression level of SaNDH6 were significantly elevated after 3 h when compared with 0 h, respectively. In conclusion, SaNDH6 was a nucleus gene encoding protein, its expression was induced by light and some hormones, and it might be involved in against some defense and stress processes in S. album.