枸杞果实发育期4CL活性和基因表达  

4CL Activity and Gene Expression during Fruit Development of Lycium chinense

作　　者：张凯敏 耿贵工[2] 乔枫 Zhang Kaimin;Geng Guigong;Qiao Feng(Key Laboratory of Medicinal Animal and Plant Resources in Qinghai Tibet Plateau,College of Life Sciences,Qinghai Normal University,Xining,810008;Academy of Agricultural and Forestry Sciences,Qinghai University,Xining,810016)

机构地区：[1]青海师范大学生命科学学院,青藏高原药用动植物资源重点实验室,西宁810008 [2]青海大学农林科学院,西宁810016

出　　处：《分子植物育种》2025年第4期1295-1304,共10页Molecular Plant Breeding

基　　金：青海省科技厅项目(2021-ZJ-729);青海省青藏高原药用动植物资源重点实验室(2020-ZJ-Y40);农业农村部农业资源环境保护项目(125A0605)共同资助。

摘　　要：本研究旨在探究枸杞果实发育期4-香豆酸:辅酶A连接酶(4CL)的活性、4CL基因相对表达量及类黄酮含量之间的关系。采用试剂盒测定枸杞果实5个发育时期(青果期,绿果期,变色期,黄熟期,红熟期)(M1~M5)的4CL酶活性,利用RT-qPCR方法测定了4CL基因的相对表达量,克隆了枸杞4CL基因(GenBank登录号:MW981504),并利用基因原核系统表达了该目的基因。结果显示:在枸杞果实发育期M1~M5,4CL酶活性和类黄酮含量都呈先下降后上升趋势,在变色期(M3)4CL酶活性和类黄酮含量最低。枸杞4CL基因的全长cDNA序列长度为2171 bp,ORF为1716 bp,编码571个氨基酸。通过BLAST对比分析,该基因编码的氨基酸与番茄等茄科植物亲缘关系最近。枸杞4CL酶活性、类黄酮含量、4CL基因表达之间呈显著性相关关系(P<0.05)。通过构建原核表达载体对枸杞4CL基因进行原核表达分析与优化,发现加入IPTG后均可诱导枸杞4CL蛋白的表达,且随着培养温度的升高,蛋白表达量逐渐增加。结果表明:可通过调节枸杞4CL活性及4CL基因表达量,进而调控类黄酮的积累。The purpose of this study is to explore the relationship between the activity of 4-coumaric acid:coenzyme A ligase(4CL),the relative expression of 4CL gene and the content of flavonoids at Lycium chinense fruit development stage.The 4CL enzyme activity of Lycium chinense fruits at five development stages(early green fruit stage,green fruit stage,discoloration stage,yellow ripening stage and red ripening stage)(M1~M5)was determined by kit.The relative gene expression of 4CL was determined by RT-qPCR.The 4CL gene of Lycium chinense was cloned(GenBank accession number:MW981504),and the target gene was expressed by gene prokaryotic system.The results showed that the enzyme activity of 4CL and flavonoid content in M1~M5 decreased first and then increased,and the enzyme activity and flavonoid content in discoloration stage(M3)were the lowest.The full-length cDNA sequence length of Lycium chinense 4CL gene was 2171 bp,ORF was 1716 bp and encoding 571 amino acids.Through BLAST comparative analysis,the amino acid encoded by this gene had the closest sequence similarity with tomato and other Solanaceae plants.There was a significant correlation between 4CL enzyme activity,flavonoid content and 4CL gene expression in Lycium chinense(P<0.05).The expression of 4CL gene was built and optimized by prokaryotic expression system.It was found that the addition of IPTG could induce the expression of 4CL protein in Lycium chinense,and the protein expression increased gradually with the increase of culture temperature.The results showed that the accumulation of flavonoids could be regulated by regulating 4CL activity and 4CL gene expression in Lycium chinense.

关 键 词：枸杞果实 4CL 基因表达 基因克隆 类黄酮含量 

分 类 号：S567.9[农业科学—中草药栽培]