栀子UGT基因家族UGT86A1和UGT85A2基因克隆与表达模式分析  

Cloning and expression pattern analysis of UGT gene family UGT86A1 and UGT85A2 in Gardenia jasminoides

作　　者：刘倩 谷振军 符潮 杨春霞[1] LIU Qian;GU Zhenjun;FU Chao;YANG Chunxia(Jiangxi Academy of Forestry,Nanchang 330032,Jiangxi,China;Central South University of Forestry and Technology,Changsha 410004,Hunan,China)

机构地区：[1]江西省林业科学院,江西南昌330032 [2]中南林业科技大学,湖南长沙410004

出　　处：《中南林业科技大学学报》2021年第11期173-182,共10页Journal of Central South University of Forestry & Technology

基　　金：国家自然科学基金项目(31760220)。

摘　　要：【目的】UDP-葡萄糖基转移酶家族基因(UGT)在植物次生代谢物的生物合成过程中起着重要的调控作用,本研究拟从栀子果实中克隆UGT基因家族成员UGT86A1和UGT85A2,对其进行生物学和表达模式分析,为阐明栀子中西红花苷合成调控机制奠定理论基础。【方法】基于栀子转录组功能注释结果,以栀子成熟果实cDNA为模板克隆目的基因,对其蛋白理化性质、信号肽和亚细胞定位预测、跨膜结构、基因结构、蛋白结构、空间结构以及不同物种中同源性对比进行分析;以栀子成熟果实DNA为模板,PCR扩增分别获得UGT86A1和UGT85A2基因组序列。利用实时荧光定量PCR技术对UGT86A1和UGT85A2基因在栀子不同部位及不同发育阶段果实的表达情况进行定量分析;采用高效液相色谱法(HPLC)测定栀子不同部位及不同发育阶段果实的西红花苷含量。【结果】克隆出的目的基因UGT86A1和UGT85A2大小分别为987、1446 bp,其编码的氨基酸数目分别为328和481个,推测蛋白分子质量(MW)分别约为36.6和53.5 kD,理论等电点(pI)分别为5.23和5.06;预测UGT86A1氨基酸序列的N端具有信号肽酶切位点,切割位点位于18和19位氨基酸之间;预测UGT86A1与UGT85A2蛋白均为叶绿体蛋白;UGT86A1蛋白在91~286位氨基酸区域和UGT85A2蛋白在207~448位氨基酸区域均存在一个保守UDPGT结构域;系统进化树分析表明UGT86A1与UGT85A2均与同为茜草科的咖啡亲缘关系最近;PCR扩增UGT86A1和UGT85A2的基因大小分别为987和1657 bp,基因结构分析表明UGT86A1基因不含内含子,UGT85A2基因含有两个外显子一个内含子;UGT86A1在栀子发育的不同阶段大致呈现递减式表达,而UGT85A2在栀子果实不同发育阶段则呈递增式表达;栀子不同发育阶段的西红花苷含量随着发育的不断成熟而增加。【结论】栀子UGT基因家族中的UGT85A2基因表达模式与西红花苷含量积累无显著相关,而UGT86A1基因表达模式与�【Objective】UDP-glucosyltransferase family genes(UGT)play an important regulatory role in the biosynthesis of plant secondary metabolites,UGT86A1 and UGT85A2,members of UGT gene family,were cloned from Gardenia jasminoides.The biological and expression patterns of UGT86A1 and UGT85A2 were analyzed to lay a theoretical foundation for elucidating the regulation mechanism of crocin biosynthesis in Gardenia jasminoides.【Method】Based on the transcriptome function annotation results of Gardenia jasminoides,the cDNA of mature fruit of Gardenia jasminoides was used as template to clone the target gene,and the physicochemical properties,Signal peptide and subcellular localization prediction,transmembrane structure,gene structure,protein structure,spatial structure and homology in different species were analyzed.The expression of UGT86A1 and UGT85A2 genes in different parts and different development stages of Gardenia jasminoides fruits were detected by real-time PCR.The content of crocin in different parts and development stages of Gardenia jasminoides was determined by high performance liquid chromatography(HPLC).【Result】The size of the cloned target genes UGT86A1 and UGT85A2 are 987 and 1446 bp,respectively,and the number of amino acids encoded are 328 and 481,respectively.It was estimated that the molecular weight(MW)of the protein are about 36.6 and 53.5 kD,respectively,and the theoretical isoelectric point(pI)are 5.23 and 5.06,respectively.It was predicted that the N-terminal of UGT86A1 amino acid sequence had signal peptide restriction site,and the cleavage site was between 18 and 19 amino acids.Phylogenetic tree analysis show that both UGT86A1 and UGT85A2 are closely related to coffee of the same rubiae family.UGT86A1 and UGT85A2 proteins were predicted to be chloroplast proteins.Phylogenetic tree analysis showed that UGT86A1 and UGT85A2 were closely related to coffee of Rubiaceae family.The size of UGT86A1 and UGT85A2 genes amplified by PCR were 987 and 1657 bp respectively,and UGT86A1 gene had no intro

关 键 词：栀子 UGT86A1 UGT85A2 克隆 西红花苷 

分 类 号：S759.95[农业科学—森林经理学]