沙糖橘S-腺苷甲硫氨酸脱羧酶基因克隆及表达分析  

Cloning and expression analysis of S-adenosylmethionine decarboxylase gene(CrSAMDC)in Shatangju

作　　者：唐文武[1] 吴秀兰 TANGWen-wu;WU Xiu-lan(College of Life Sciences,Zhaoqing University,Zhaoqiing,Guangdong 526061,China;College of Food and Pharmaceutical Engineering,Zhaoqing University,Zhaoqing,Guangdong 526061,China)

机构地区：[1]肇庆学院生命科学学院,广东肇庆526061 [2]肇庆学院食品与药品工程学院,广东肇庆526061

出　　处：《南方农业学报》2020年第6期1369-1376,共8页Journal of Southern Agriculture

基　　金：广东省科技计划项目(2014A020208144);肇庆市科技计划项目(2015B010201001)。

摘　　要：【目的】克隆沙糖橘S-腺苷甲硫氨酸脱羧酶基因(CrSAMDC),并明确其在不同组织和干旱胁迫下的表达特征,为开展SAMDC基因分子机理研究提供理论依据,同时为柑橘抗逆分子育种提供候选基因。【方法】以6年生沙糖橘为材料,利用RT-PCR克隆CrSAMDC基因并进行生物信息学分析,以实时荧光定量PCR检测CrSAMDC基因在不同组织和干旱胁迫下的表达谱,并开展原核诱导外源蛋白表达及Southern杂交分析。【结果】从沙糖橘嫩叶克隆获得的CrSAMDC基因cDNA序列全长1736 bp,含有1个1131 bp的开放阅读框(ORF),编码376个氨基酸残基;CrSAMDC蛋白相对分子量为40.69 kD,理论等电点(pI)为5.27,为亲水性蛋白,其二级结构以α-螺旋和无规则卷曲为主,分别占32.98%和42.02%,β-转角仅占6.91%,延伸链占18.09%;与克里曼丁橘(Citrus clementina,XP_024047984.1)和甜橙(C.sinensis,KDO62315.1)的SAMDC蛋白亲缘关系较近,其氨基酸序列同源性分别为99.5%和97.3%;具有LSE-SSLF的酶原剪切位点结构域及与SAMDC蛋白降解相关的PEST结构域(TVHITPED-GFSYASYE)。CrSAMDC基因在沙糖橘的叶、花、果肉和果皮中均有表达,且在分裂旺盛的嫩叶和花器官中高表达;干旱胁迫下,CrSAMDC基因表达量呈先上升后下降的变化趋势,以干旱胁迫后24 h的相对表达量最高。CrSAMDC基因在沙糖橘基因组中以单拷贝形式存在,且通过构建原核表达载体能成功诱导表达获得CrSAMDC融合蛋白。【结论】CrSAMDC基因在沙糖橘中的表达具有组织特异性,且以单拷贝存在,在干旱胁迫下呈上调表达趋势,即参与干旱胁迫响应的多胺代谢调控。【Objective】The S-adenosylmethionine decarboxylase gene(CrSAMDC)was cloned from Shatangju,and the expression characteristics of the CrSAMDC gene was analyzed in different tissues and under drought stress.This provided basis for further molecular mechanism of SAMDC gene and provided candidate genes for Citrus stress resistant molecular breeding.【Method】CrSAMDC gene was cloned by using reverse transcriptional PCR(RT-PCR)with six-year Shatangju as materials.Bioinformatics was conducted as well.Real-time fluorescence quantitative PCR(qRT-PCR)method was adopted to study the expressions of CrSAMDC gene in different tissues and under drought stress.Prokaryotic induction exogenous protein expression and Southern blot analysis were conducted.【Result】The cDNA full-length sequence of CrSAMDC gene cloned fron Shatangju new leaves was 1736 bp,with an open reading frame(ORF)of 1131 bp,encoding 376 amino acids.CrSAMDC protein relative molecular weight was 40.69 kD,theoreticalisoelectric point(pI)was 5.27.It was a hydrophilic protein.Its secondary structure was mainly consisted ofα-helix(32.98%)and random coil(42.02%),β-turn only accounted for 6.91%,and extended chain accounted for 18.09%.It had the close genetic relationship with SAMDC protein of Citrus clementina(XP_024047984.1)and C.sinensis(KDO62315.1),their amino acid sequence homology were 99.5%and 97.3%.It had LSE-SSLF prozyme cleavage site domain and PEST domains related to SAMDC protein degradation(TVHITPED-GFSYASYE).CrSAMDC gene was expressed in leaves,flowers,flesh and peel of Shatangju,and was highly expressed in young leaves and flowers with vigorous division.Under drought stress,the expression level of CrSAMDC gene first increased and then decreased,and the highest expression level was reached at 24 h.CrSAMDC gene exists as a single copy,and CrSAMDC fusion protein could be obtained through induction of prokaryotic expression vector.【Conclusion】The expression of CrSAMDC gene in Shatangju has tissue specificity,and exists as single copy.It upregulate

关 键 词：沙糖橘 CrSAMDC基因 多胺 干旱胁迫 原核表达 SOUTHERN杂交 

分 类 号：S666.2[农业科学—果树学]