摘要
水杨酸(salicylic acid,SA)是植物防御反应过程中的关键信号分子,异分支酸途径是SA合成的主要途径。异分支酸合酶(isochorismate synthase,ICS)是该途径的限速酶,催化分支酸变构为异分支酸,再经丙酮酸裂解酶的催化分解形成水杨酸。本研究以宁夏枸杞(Lycium barbarum)叶片为材料,利用cDNA末端快速扩增(rapid amplification of cDNAends,RACE)技术克隆枸杞ICS基因,利用Blastn和Blastp进行核酸和氨基酸序列相似性分析同时构建进化树;利用ExPaSy在线分析预测蛋白质二级结构与构建3D结构模型;利用荧光定量qPCR测定LbICS在枸杞不同组织器官中的表达特性,进一步分析蚜虫取食枸杞叶片过程中LbICS表达谱与水杨酸合成的关系。结果显示:枸杞LbICS基因(GenBank登录号为MK457455)开放阅读框1731 bp,编码577个氨基酸,具有多个磷酸化位点,构建的蛋白质3D模型为一个紧凑的球状结构。经Signal P4.0 Server等(在线软件)分析发现LbICS蛋白含有典型的保守分支酸结合结构域,N端含有一条37个氨基酸残基组成的叶绿体跨膜信号肽,与辣椒(Capsicum annuum XM_016722852)ICS相似度最高,其次与烟草(Nicotiana sylvestris XM_019374845)ICS、牵牛花(Ipomoea nil XM_019342500)ICS、橙子(Citrus sinensis XM_00647-6586)ICS等亲缘关系较近。RT-qPCR结果显示,LbICS在枸杞不同的器官中表达具有组织特异性,受外源SA诱导表达,在叶中的表达量最高,茎次之,根中表达量最低;枸杞叶片被蚜虫取食后1~3 d LbICS表达逐渐升高,3 d时达到最大值,取食后5~7 d逐渐降低,其表达变化模式与蚜虫取食后叶片SA含量变化趋势一致。以上结果表明蚜虫取食下LbICS调控枸杞SA合成,研究结果对于阐明SA在枸杞中的合成途径及枸杞LbICS的生理功能具有重要意义。
Salicylic acid(SA)is a key signaling molecule in the process of plant defense reaction.The ICS pathway is the main pathway for SA synthesis,and isochorismate synthase(ICS)is the rate-limiting enzyme in the synthesis pathway of isochoridate.It catalyzes the allosteric transformation of chorismate to isochoridate,which is then decomposed by pyruvatelyase to form salicylic acid.In this study,Lycium barbarum leaves were used as materials.RACE(rapid amplification of c DNA ends)technology was used to clone the wolfberry ICS gene,and Blastn and Blastp were used to analyze the similarity of nucleic acid and amino acid sequences and build an evolutionary tree.ExPaSy online analysis was used to predict protein secondary structure and construct a 3D structural model.Using fluorescence quantitative qPCR to determine the expression characteristics of LbICS in different tissues and organs,and further analyze the relationship between the expression profile of LbICS and the synthesis of salicylic acid during aphids feeding on the leaves.The results showed that the LbICS(GenBank accession number MK457455)has an open reading frame of 1731 bp,encodes 577 amino acids,and has multiple phosphorylation sites.The constructed protein 3 D model is a compact spherical structure.Analysis by Signal P4.0 Server and others(online software)found that the LbICS protein contains a typical conserved chorionic acid binding domain,and the N-terminus contains a 37-amino acid residue chloroplast transmembrane signal peptide,with the highest similarity to pepper(Capsicum annuum XM_016722852)ICS,followed by tobacco(Nicotiana sylvestris XM_019374845)ICS,morning glory(Ipomoea nil XM_019342500)ICS,orange(Citrus sinensis XM_006476586)ICS and other close relationships.Real time PCR results showed that the expression of LbICS in different organs of Lycium barbarum was tissue-specific,and the expression level in the leaves reached the highest value.Real time qPCR results showed that the expression of LbICS is tissue-specific,with the highest expression in leaves,followed by stems,and lowest in roots.The expression of LbICS gradually increased at 1~3 days after aphid feeding and reached the maximum at 3 days the value gradually decreased afterwards,which was consistent with the change trend of SA content in the leaves of aphids after feeding.The above results showed that LbICS of wolfberry regulated the synthesis of wolfberry SA under aphids feeding.The results of the study are of great significance for elucidating the synthetic pathway of SA in Lycium barbarum and the physiological function of LbICS.
作者
王丽娟
徐惠娟
杨亚珺
兰卉泽
王婷婷
马婷
高媛媛
Wang Lijuan;Xu Huijuan;Yang Yajun;Lan Huize;Wang Tingting;Ma Ting;Gao Yuanyuan(College of Basic Medical Science,Ningxia Medical University,Yinchuan,750004;College of Life Science,Ningxia University,Yinchuan,750004)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第7期2801-2809,共9页
Genomics and Applied Biology
基金
宁夏自然科学基金(2021AAC03145)
宁夏医科大学基金(NYD2020002)项目共同资助
关键词
枸杞
异分支酸合酶
表达模式
水杨酸
蚜虫取食
Lycium barbarum
Isochorismate synthase
Expression pattern
Salicylic acid
Aphid feeding
