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

作　　者：闫海锋 吕金凤 熊发前 丘立杭 周慧文 陈兴隆 马国华[6] YAN Haifeng;L Jinfeng;XIONG Faqian;QIU Lihang;ZHOU Huiwen;CHEN Xinglong;MA Guohua(Sugarcane Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,China;Key Laboratory of Sugarcane Biotechnology and Genetic Improvement,Ministry of Agriculture and Rural Affairs,Nanning 530007,China;Key Laboratory of Guangxi Sugarcane Genetic Improvement,Nanning 530007,China;Guangxi Forestry Group Guiqinlin Pulp Paper Co.Ltd.,Nanning 530012,China;Agriculture College of Guangxi University,Nanning 530004,China;South China Botanical Garden,Chinese Academy of Sciences,Guangzhou 510650,China)

机构地区：[1]广西壮族自治区农业科学院甘蔗研究所,南宁530007 [2]农业农村部广西甘蔗生物技术与遗传改良重点实验室,南宁530007 [3]广西甘蔗遗传改良重点实验室,南宁530007 [4]广西林业集团桂钦林浆纸有限公司,南宁530012 [5]广西大学农学院,南宁530004 [6]中国科学院华南植物园,广州510650

出　　处：《广西植物》2024年第5期951-960,共10页Guihaia

基　　金：国家自然科学基金(32060358);广东省重点科技项目(2015B020231008);广西自然科学基金(2019GXNSFAA185005)。

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SaNDH 6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,SaNDH 6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(GA 3)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)SaNDH 6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GA 3分别处理檀香愈伤组织后,与处理前(0 h)相比,SaNDH 6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。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 SaNDH 6 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 SaNDH 6 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(GA 3)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 SaNDH 6 promoter,and among which,ERF transcription factor was the most(up to 40 TFs).(5)SaNDH 6 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 GA 3 treatments,the expression level of SaNDH 6 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

关 键 词：檀香 叶绿体 NDH脱氢酶 亚细胞定位 表达调控 

分 类 号：Q943[生物学—植物学]