薄壳山核桃CiSPL2基因克隆、亚细胞定位及表达分析  

Cloning,subcellular localization and expression analysis of CiSPL2 gene in pecan

作　　者：王敏[1,2] 席东[3] 莫正海[1] 张仕杰[1] 朱灿灿 WANG Min;XI Dong;MO Zheng-hai;ZHANG Shi-jie;ZHU Can-can(Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing,Jiangsu 210095,China;Horticul-ture Research Institute,Shanghai Academy of Agricultural Sciences,Shanghai 201403,China;Qingpu District Agricultural Technology Extension Service Center in Shanghai,Shanghai 201700,China)

机构地区：[1]江苏省中国科学院植物研究所,江苏南京210095 [2]上海市农业科学院园艺研究所,上海201403 [3]上海市青浦区农业技术推广服务中心,上海201700

出　　处：《南方农业学报》2024年第1期47-56,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32001350);江苏省自然科学基金项目(BK20210166)。

摘　　要：【目的】克隆薄壳山核桃SQUAMOSA启动子结合蛋白(SPL)转录因子基因CiSPL2,并对其进行亚细胞定位及表达分析,为探究该基因在薄壳山核桃雌花发育过程的分子机制提供理论依据。【方法】根据薄壳山核桃基因组数据,采用RT-PCR方法克隆CiSPL2基因编码区(CDS)序列,利用生物信息学软件分析其序列特征和蛋白理化性质,构建pCAMBIA1300-CiSPL2-GFP融合表达载体,瞬时转化烟草后观察荧光信号确定亚细胞定位情况。基于转录组数据和实时荧光定量PCR检测CiSPL2基因在不同组织及不同雌花芽分化时期的表达模式。【结果】薄壳山核桃CiSPL2基因CDS长度为1398 bp,共编码465个氨基酸残基,相对分子量为51.78 kD,理论等电点(p I)为8.28,不稳定系数为49.89,脂肪酸氨基酸指数为64.62,平均亲水性平均值(GRAVY)为-0.617,为不稳定的亲水性蛋白,含有SBP结构域(位于第183~257位氨基酸),亚细胞定位于细胞核。CiSPL2蛋白的二级结构主要由α-螺旋(17.42%)、延伸链(13.55%)、β-转角(1.72%)和无规则卷曲(67.31%)组成。薄壳山核桃CiSPL2蛋白与核桃JrSPL2蛋白氨基酸序列的相似性最高,为95.05%,其次是光皮桦BlSPL2蛋白,相似性为70.83%。CiSPL2蛋白与核桃JrSPL2蛋白亲缘关系最近。CiSPL2基因启动子中含有植物激素响应、干旱胁迫响应和分生组织表达元件。CiSPL2基因在雌花和果实中的表达水平显著高于其他组织(P<0.05),在雌花芽分化各阶段均有表达,在雌花序形成期表达水平最高。【结论】CiSPL2基因含有SPL转录因子家族典型的SBP保守结构域,推测其参与调控薄壳山核桃雌花芽分化和发育过程。【Objective】This study conducted the gene cloning of the pecan SQUAMOSA promoter binding protein(SPL)transcription factor gene CiSPL2,subcellular localization and expression analysis were also conducted,in order to provide a theoretical basis for exploring the molecular mechanism of this gene during the female flower bud differentiation in pecan.【Method】The coding region(CDS)sequence of CiSPL2 gene was cloned using RT-PCR based on the pecan ge-nome data,and the bioinformatics software was used to analyze its sequence characteristics and protein physical and chemical properties.The pCAMBIA1300-CiSPL2-GFP fusion expression vector was constructed,and subcellular localiza-tion was determined by observing fluorescence signals after transient transformation of tobacco.The expression patterns of CiSPL2 gene in different tissues and female flower bud differentiation stages were studied based on the transcriptome data and real-time fluorescence quantitative PCR.【Result】The CDS length of the CiSPL2 gene in pecan was 1398 bp,enco-ding a total of 465 amino acids residues with a relative molecular weight of 51.78 kD.The theoretical isoelectric point(pI)was 8.28,the instability coefficient was 49.89,the fatty acid amino acid index was 64.62,and the grand average of hydropathicity(GRAVY)was-0.617,indicating the protein was an unstable hydrophilic protein.The CiSPL2 protein se-quence contained an SBP domain between the 183rd and 257th amino acids.Subcellular localization showed that CiSPL2 protein was located in the nucleus.The secondary structure of CiSPL protein was mainly composed ofα-helix(17.42%),extended chain(13.55%),β-turn(1.72%)and random coil(67.31%).CiSPL2 protein in pecan had the highest similarity with JrSPL2 protein in walnut(95.05%),followed by Betula platyphylla BlSPL2 protein with a similarity of 70.83%.CiSPL2 protein had the closest genetic relationship with the walnut JrSPL2 protein.The CiSPL2 gene promoter contained plant hormone response,drought stress response,and meristem expression elements.T

关 键 词：薄壳山核桃 CiSPL2基因 系统发育分析 亚细胞定位 基因表达 

分 类 号：S664.1[农业科学—果树学]