摘要
【目的】磷酸乙醇胺N-甲基转移酶(phosphoethanolamine N-methyltransferse,PEAMT)是植物磷酸胆碱合成的关键酶,而磷酸胆碱是可以增强植物抗性的甘氨酸甜菜碱合成底物胆碱的前体。通过对陆地棉磷酸乙醇胺N-甲基转移酶基因(GhPEAMT1)的克隆、表达模式分析,以及功能验证,探究GhPEAMT1在陆地棉响应盐胁迫中的生物学功能,为棉花耐盐品种的选育提供基因资源。【方法】根据转录组测序数据分析,最终确定GhPEAMT1为耐盐候选基因;通过聚合酶链式反应(PCR)扩增目的基因;通过生物信息学分析基因结构特征、预测蛋白质相对分子质量以及进化关系;利用荧光定量PCR(qRT-PCR)分析耐盐棉花品种早熟长绒7号和感盐棉花品种南丹巴地大花在NaCl胁迫后不同时间点不同组织的表达特征;构建亚细胞定位载体,进行烟草的瞬时转化,确定蛋白质在细胞中的位置;构建基因超表达载体,通过花序浸染法转化拟南芥,分析转基因拟南芥种子在盐胁迫下的萌发率和转基因拟南芥主根长度;利用病毒诱导基因沉默技术(virus induced gene silencing,VIGS)对早熟长绒7号棉花品种进行目的基因沉默,提取棉花叶片的RNA进行实时荧光定量用以检测基因沉默效率,随后用40 g·L^-1的NaCl溶液的处理对照棉花植株(CK)、注射TRV2:00的棉花植株、GhPEAMT1A和Gh PEAMT1D沉默成功的棉花植株,测定棉花叶片过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)的活性。【结果】克隆获得GhPEAMT1的2个同源基因GhPEAMT1A和GhPEAMT1D,2个基因的CDS全长分别为1488和1485 bp;构建进化树发现,GhPEAMT1与海岛棉同源基因的亲缘关系最近,而与其他物种相比,与可可同源性最高;盐胁迫条件下,GhPEAMT1A和GhPEAMT1D在耐盐品种早熟长绒7号叶片和根中的表达量显著高于感盐品种南丹巴地大花。亚细胞定位显示该基因位于细胞质中;超表达转基因拟南芥后,�
【Objective】Phosphoethanolamine-N-methyltransferase(PEAMT)is the key enzyme in the synthesis of plant phosphocholine,which is the precursor of choline,a glycine betaine synthesis substrate that can enhance plant resistance.The biological function of GhPEAMT1 gene in response to salt stress in upland cotton was studied by cloning,expression pattern analysis and functional verification,so as to provide gene resources for breeding salt tolerant cotton varieties.【Method】According to the screening of transcriptome sequencing data,GhPEAMT1 gene was selected as a salt-tolerant candidate gene.The target gene was amplified by polymerase chain reaction(PCR).Gene structure characteristics predict protein relative molecular mass and evolutionary relationship were analyzed by bioinformatics;Salt-tolerant cotton variety Earlistable 7 and salt-sensitive cotton genotype Nandanbadidahua were treated with 200 mmol·L^-1 NaCl solution,and cotton leaves and roots were taken for fluorescence quantitative PCR(qRT-PCR)to analyze the expression characteristics in tissues.Subcellular location vector was constructed for transient transformation of tobacco to determine the location of protein in the cell.The gene overexpression vector was constructed,and Arabidopsis thaliana was transformed by inflorescence infection method,and the germination rate and taproot length of transgenic Arabidopsis under salt stress were analyzed.Virus induced gene silencing(VIGS)technology was used on Earlistable 7 to silence the target gene and the efficiency of gene silencing was verified by real-time fluorescence quantitative analysis of the cotton leaves.Then the enzyme activities of catalase(CAT)and glutathione peroxidase(GPX)in the leaves of cotton were measured.【Result】Two homologous genes,GhPEAMT1 A and GhPEAMT1 D,were cloned and the CDS lengths were 1488 bp and 1485 bp,respectively.The phylogenetic tree showed that GhPEAMT1 had the closest genetic relationship with the Sea island cotton,and had the higher homology with Cocoa compared with ot
作者
王娜
赵资博
高琼
何守朴
马晨辉
彭振
杜雄明
WANG Na;ZHAO ZiBo;GAO Qiong;HE ShouPu;MA ChenHui;PENG Zhen;DU XiongMing(Institute of Cotton Research,Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology,Anyang 455000,Henan;School of Agricultural Sciences,Zhengzhou University/Zhengzhou Research Base,State Key Laboratory of Cotton Biology,Zhengzhou 450001)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第2期248-260,共13页
Scientia Agricultura Sinica
基金
国家重点研发计划(2017YFD0101600)
中央级公益性科研院所基本科研业务费专项(161012018004)。
