杉木ATP合酶基因的克隆及其在逆境下的表达分析  

Cloning of ATPase Gene of Cunninghamia lanceolata and Its Expression in Response to Different Stress

作　　者：汪佳琪 戴嘉豪 陈家琛 林莉莉 曹光球[1,2,3] 曹世江 WANG Jiaqi;DAI Jiahao;CHEN Jiachen;LIN Lili;CAO Guangqiu;CAO Shijiang(College of Forestry,Fujian Agriculture and Forestry University,Fuzhou 350002,China;University Key Laboratory of Forest Stress Physiology,Ecology and Molecular Biology of Fujian Province,Fuzhou 350002,China;Chinese Fir Engineering Technology Research Center of National Forestry and Grassland Administration,Fuzhou 350002,China;Huachuan Technology Co.,Ltd,Fuzhou 350008,China)

机构地区：[1]福建农林大学林学院,福建福州350002 [2]林木逆境生理生态及分子生物学福建省高校重点实验室,福建福州350002 [3]国家林业和草原杉木工程技术研究中心,福建福州350002 [4]华川技术有限公司,福建福州350008

出　　处：《江西农业大学学报》2022年第2期271-279,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31600228)。

摘　　要：【目的】为解析杉木ATP合酶复合亚基基因(ClF0F1-ATP)在抵抗逆境胁迫中的功能,研究克隆了杉木ClF0F1-ATP基因,并分析其在不同逆境中的表达特征,以期为阐明杉木ClF0F1-ATP基因在杉木生长发育和响应干旱、温度以及盐胁迫逆境过程中的分子机制提供科学的理论依据。【方法】以第三代杉木种子为试验材料,运用RT-PCR技术克隆获得了一个杉木ATP合酶基因,命名为ClF0F1-ATP,并采用了生物信息学技术与亚细胞定位研究对该基因的功能进行了分析和预测,利用实时荧光定量PCR(qRT-PCR)分析了该基因在低强度干旱、高强度干旱、低盐、高盐、低温以及高温胁迫下的表达模式。【结果】对ClF0F1-ATP基因的编码的氨基酸序列进行的预测分析结果显示,杉木ClF0F1-ATP基因全长618 bp,共编码205个氨基酸,杉木ClF0F1-ATP蛋白的化学分子式为C_(983)H_(1561)N_(259)O_(291)S_(6),蛋白相对分子量为21856.13 Da,理论等电点为6.20。蛋白质理化性质和结构分析结果显示ClF0F1-ATP蛋白理化性质稳定,带正电荷,属于疏水性蛋白,与线粒体ATP合酶亚基的结构相似程度较高。蛋白的亚细胞定位和同源序列对比结果显示该蛋白定位于细胞核,与卷柏(s.m184168)的亲缘关系最近,相似度为94%。实时荧光定量PCR结果分析表明杉木ClF0F1-ATP基因在低强度干旱、低盐、低温胁迫下6,12,24,48 h后的表达量均高于对照组,且随时间增加呈现先上升后下降的趋势,而在高强度干旱、高盐、高温胁迫下48 h内的表达量均低于对照组,随时间增加呈现下降趋势。【结论】ClF0F1-ATP基因在杉木中的表达受不同逆境胁迫的诱导,其中低强度干旱、低盐、低温胁迫诱导其上调表达,而高强度干旱、高盐、高温胁迫诱导其下调表达,由此推测该基因可能通过参与杉木的逆境胁迫响应,从而调控杉木的生长发育,但其中具体的作用机理还有待进一步研究。在后续[Objective]In order to analyze the function of Chinese fir ATP synthase complex subunit(CLF0F1-ATP)gene in resistance to stress,the ClF0F1-ATP gene was cloned and its expression characteris‐tics in different stresses were analyzed.This study aims to provide a scientific theoretical basis for elucidating the molecular mechanism of ClF0F1-ATP gene in the growth and development of Cunninghamia lanceolata and its response to drought,temperature and salt stress.[Method]The seeds of the third generation of Cunninghamia lanceolata were used as experimental materials.ATP synthase gene of Chinese fir was cloned by RT-PCR and named ClF0F1-ATP,and its function was analyzed and predicted by bioinformatics technology and subcellular localization.Real-time fluorescence quantitative PCR(qRT-PCR)was used to analyze the expres‐sion patterns of ClF0F1-ATP gene under low concentration drought stress,high concentration drought stress,low salt stress,high salt stress,low temperature stress and high temperature stress.[Result]The prediction anal‐ysis of amino acid sequence of CLF0F1-ATP gene showed that the total length of ClF0F1-ATP gene was 618 bp,which encoded 205 amino acids.The molecular formula of CLF0F1-ATP protein of Cunninghamia lcunninghamia was C_(983)H_(1561)N_(259)O_(291)S_(6).The relative molecular weight of the protein was 21856.13 Da and the theoretical isoelectric point was 6.20.The physicochemical properties and structure analysis of ClF0F1-ATP protein indi‐cated that it was a hydrophobic protein with stable physicochemical properties and a high degree of structural similarity with mitochondrial ATP synthase subunit.The results of subcellular localization and homologous se‐quence comparison showed that the protein was located in the nucleus,and it was closely related to selaginella(s.m184168)with 94%similarity.The results of real-time quantitative PCR showed that the expression level of CLF0F1-ATP gene in Cunninghamia lcunninghamia was higher than that in the control group after 6 h,12 h,24 h and 48 h under

关 键 词：杉木 基因克隆 逆境 表达分析 

分 类 号：Q945.11[生物学—植物学]