灰毡毛忍冬与忍冬HQT2基因的克隆及表达分析  

Cloning and expressions of HQT2 genes in Lonicera macranthoides Hand.-Mazz.and Lonicera japonica Thunb.

作　　者：刘湘丹[1,2,3] 陈勋 龙雨青 王志辉 刘笑蓉[1,2] 王朝晖 童巧珍[1,2,3] 周日宝 LIU Xiangdan;CHEN Xun;LONG Yuqing;WANG Zhihui;LIU Xiaorong;WANG Zhaohui;TONG Qiaozhen;ZHOU Ribao(School of Pharmacy,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;Key Laboratory for the Germplasm Resources and Standardized Planting of Hunan's Bulk Authentic Medicinal Materials,Changsha,Hunan 410208,China;Hunan Provincial Key Laboratory of Chinese Medicinal Modernization,Changsha,Hunan 410208,China;Nanhua Hospital of University of South China,Hengyang,Hunan 421002,China)

机构地区：[1]湖南中医药大学药学院,湖南长沙410208 [2]湘产大宗道地药材种质资源及规范化种植重点研究室,湖南长沙410208 [3]湖南省普通高等学校中药现代化研究重点实验室,湖南长沙410208 [4]南华大学附属南华医院,湖南衡阳421002

出　　处：《湖南中医药大学学报》2023年第7期1215-1224,共10页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金项目(81673546,81203007);湖南省自然科学基金项目(2021JJ30497,2021JJ30515);国家现代农业产业技术体系建设专项项目(CARS-21);湖南省现代农业产业技术体系建设专项项目;湖南省大学生创新训练项目(202104080117);湖南中医药大学研究生创新训练项目(2021CX80,2022CX79);2020年湖南省一流专业建设点:中药资源与开发。

摘　　要：目的分别克隆灰毡毛忍冬与忍冬HQT2基因全长序列,并进行生物信息学和表达量分析,以揭示HQT2在灰毡毛忍冬与忍冬绿原酸生物合成中的可能功能。方法多糖多酚试剂盒法分别提取灰毡毛忍冬与忍冬花总RNA,通过RT-PCR和RACE技术克隆HQT2基因的全长c DNA序列,运用相关软件对该基因序列进行生物信息学分析;利用实时荧光定量PCR技术(qRT-PCR)分别测定灰毡毛忍冬与忍冬茎、叶及不同花期花中相关基因的相对表达量;采用HPLC测定绿原酸含量并结合表达量做相关性分析。结果克隆得到LmHQT2(MH196564)和LjHQT2(MK294639),其开放阅读框长度均为1296 bp,编码431个氨基酸,生物信息学分析预测为亲水性蛋白,可能定位于细胞质中,属于氯霉素乙酰转移酶样结构域超家族;qRT-PCR结果显示,HQT2基因具有组织特异性,在灰毡毛忍冬与忍冬茎、叶及不同花期花器官中表达存在差异;绿原酸含量与基因相对表达量之间呈现一定的相关性。结论本研究成功克隆了灰毡毛忍冬与忍冬HQT2基因并探索其在不同器官中的表达模式,推测HQT2基因在灰毡毛忍冬与忍冬的绿原酸生物合成途径中发挥不同功能,为进一步研究该基因的功能及探究灰毡毛忍冬和忍冬绿原酸生物合成调节机制提供了研究基础。Objective To reveal the possible functions of HQT2 gene in the chlorogenic acid biosynthesis of Lonicera macranthoides Hand.-Mazz.(L.macranthoides)and Lonicera japonica Thunb.(L.japonica)by cloning the full-length sequences of HQT2 genes from L.macranthoides and L.japonica respectively for bioinformatics and expression analysis.Methods The total RNAs of L.macranthoides and L.japonica were extracted respectively using Biospin Polysaccharide Polyphenol Plant Total RNA Extraction Kit.The full-length cDNA sequences of HQT2 genes were cloned by RT-PCR and RACE,and bioinformatics analysis was performed on them with relevant softwares.Real-time fluorescence quantitative PRC(qRT-PCR) was used to determine the relative expression levels of the genes in the stems and leaves, as well as the flowers at different flowering stages of L. macranthoides and L. japonica respectively. The content of chlorogenic acid was determined by HPLC and the correlation between it and the relative expression level of HQT2 gene was analyzed. Results The full-length cDNA sequences of LmHQT2 gene (MH196564) and LjHQT2 gene (MK294639) were cloned successfully, each of which contained a 1296 bp open reading frame (ORF) and encoded 431 amino acids. Bioinformatics predictive analysis indicated that the proteins encoded by the two genes were hydrophili, possibly located in the cytoplasm, belonging to the chloramphenicol acetyltransferase-like domain superfamily. qRT-PCR showed that HQT2 gene was tissue-specific, and its expression levels were different in the stems and leaves, as well as the floral organs at different flowering stages of L. macranthoides and L. japonica. There was a certain correlation between chlorogenic acid content and the relative expression level of this gene. Conclusion This study cloned HQT2 genes of L. macranthoides and L. japonica successfully and analyzed their expressions in the different plant organs. It is speculated that HQT2 gene plays different functions in the chlorogenic acid biosynthesis pathways of L. macranthoides

关 键 词：灰毡毛忍冬 忍冬 HQT2 基因克隆 生物信息学分析 表达分析 

分 类 号：R282.2[医药卫生—中药学]