烟草NtDEAH1基因的克隆、生信分析及功能验证  

Cloning,bioinformatics analysis and functional verification of NtDEAH1 gene in Nicotiana tabacum

作　　者：王余 吴贵芝 崔帅 唐琴 谭永言 李长远 唐华江 杨豪 黄莺 刘仁祥 WANG Yu;WU Gui-zhi;CUI Shuai;TANG Qin;TAN Yong-yan;LI Chang-yuan;TANG Hua-jiang;YANG Hao;HUANG Ying;LIU Ren-xiang(College of Tobacco,Guizhou University/Guizhou Key Laboratory of Tobacco Quality Research,Guiyang 550025,China)

机构地区：[1]贵州大学烟草学院/贵州省烟草品质研究重点实验室,贵阳550025

出　　处：《西南农业学报》2025年第1期23-30,共8页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(32060510);贵州省基础研究计划自然科学项目(黔科合基础-ZK[2023]116);贵州省烟草公司项目(2022XM02)。

摘　　要：【目的】探明烟草NtDEAH1基因的特异性表达、逆境胁迫响应模式及其对烟草叶片数的影响,为进一步研究NtDEAH1基因调控烟草叶片数的分子机制提供理论依据。【方法】以烟草K326、韭菜坪2号和NC82为材料,利用PCR克隆NtDEAH1基因,运用在线网站进行生物信息学分析,使用RT-qPCR方法分析该基因在不同器官(根、茎、叶)、不同发育时期(团棵-现蕾期)和不同胁迫(干旱、盐、高温和低温胁迫)处理下的表达模式,利用遗传转化方法将过表达和敲除载体通过农杆菌转到烟草叶片中创制突变体,研究该基因对烟草叶片数的影响。【结果】NtDEAH1基因是RNA解旋酶基因家族成员之一,CDS开放阅读框为2829 bp,编码942个氨基酸,分子质量为106.579 kDa;NtDEAH1蛋白定位于细胞核,属于不稳定亲水性蛋白质;NtDEAH1基因在烟株生长发育进程中的表达量呈先增后降趋势,在花芽分化期前最高,NC82相对表达量为27.45,韭菜坪2号的相对表达量为13.56;NtDEAH1基因响应干旱、高温及低温胁迫,相对表达量随处理时间延长呈先升后降趋势,12 h时叶中的相对表达量最高,干旱胁迫为7.04,高温胁迫为8.42,低温胁迫为16.65;NtDEAH1基因敲除突变体的叶片数(韭菜坪2号33.33片,NC8226.67片)显著高于野生型(韭菜坪2号27.67片,NC8219.67片),过表达株系的叶片数(韭菜坪2号19.67片,NC8215.67片)显著少于野生型(韭菜坪2号27.67片,NC8219.67片)。【结论】本研究克隆获得NtDEAH1基因,其属于RNA解旋酶基因家族,能响应干旱、高温和低温逆境胁迫,具有调控烟株生长发育和叶片数的功能。【Objective】The specific expression and stress response patterns of NtDEAH1 gene in Nicotiana tabacum,and the effect on leaf number of tobacco plants were explored to provide a theoretical basis for further studying the molecular mechanism of NtDEAH1 gene in regulating the leaf number of N.tabacum.【Method】The NtDEAH1 gene was cloned from tobacco varieties K326,JCP2 and NC82 by PCR and the bioinformatics was analyzed by online websites.The expression patterns of NtDEAH1 gene in different organs(root,stem and leaf)of tobacco plants,at different developing stages(resettling stage-squaring stage)and under different stresses(drought,salt,high temperature and low temperature)were analyzed by RT-QCPR.The overexpression and knockout vectors were transferred to tobacco leaves by Agrobacterium tumefaciens to study the effects of NtDEAH1 gene on leaf number of tobacco plants.【Result】NtDEAH1 gene was a member of RNA helicase gene family.The open reading frame of CDS was 2829 bp with encoding 942 amino acids and its molecular weight was 106.579 kDa.NtDEAH1 protein located on the cell nucleus belonged to unstable hydrophilic protein.The expression of NtDEAH1 gene in tobacco plants showed a first increase and then decrease trend during the growing development process and was the highest before flower bud differentiation stage.The relative expression of NC82 and Jiucaiping 2 was 27.45 and 13.56 respectively.The relative expression of NtDEAH1 gene presented a first up and then down trend with extension of treatment time under drought,high temperature and low temperature stress.The relative expression of NtDEAH1 gene in tobacco leaves under drought treatment for 12 hours,high temperature treatment for 12 hours and low temperature for 12 hours reached 7.04,8.42 and 16.65,separately.The leaf number of NtDEAH1 knockout mutants of Jiucaiping 2 and NC82 was 33.33 and 26.67,significantly higher than 27.67 and 19.67 of Jiucaiping 2 and NC82 with wild type respectively.The leaf number of NtDEAH1 overexpression lines of Jiucaipi

关 键 词：烟草 NtDEAH1 叶片数 生物信息学分析 功能验证 

分 类 号：S572[农业科学—烟草工业]