生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 256-267.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0194

• 研究报告 • 上一篇    下一篇

灰毡毛忍冬UGTPg17、UGTPg36基因克隆及功能研究

杨敏1(), 龙雨青1, 曾娟1, 曾梅1, 周新茹1, 王玲1, 付学森1, 周日宝1,2,3(), 刘湘丹1,2,3()   

  1. 1.湖南中医药大学药学院,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,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,长沙 410208
  • 收稿日期:2023-03-06 出版日期:2023-10-26 发布日期:2023-11-28
  • 通讯作者: 周日宝,男,博士,教授,博士生导师,研究方向:中药资源与品质评价;E-mail: 1057323510@qq.com
    刘湘丹,女,博士,副教授,硕士生导师,研究方向:中药资源与品质评价;E-mail: paeonia_dd@126.com
  • 作者简介:杨敏,女,硕士研究生,研究方向:中药资源与质量;E-mail: 2574467409@qq.com
  • 基金资助:
    国家现代农业产业技术体系(CARS-21);湖南省自然科学基金(2021JJ30497);湖南省自然科学基金(2021JJ30515);2022湖南省中医药科研计划项目(D2022134);2021湖南省教育厅科学研究项目(21C0245);2020年湖南省一流专业建设点:中药资源与开发,湖南中医药大学中药学一流学科项目(校行科字【2018】3号)

Cloning and Function Analysis of Gene UGTPg17 and UGTPg36 in Lonicera macranthoides

YANG Min1(), LONG Yu-qing1, ZENG Juan1, ZENG Mei1, ZHOU Xin-ru1, WANG Ling1, FU Xue-sen1, ZHOU Ri-bao1,2,3(), LIU Xiang-dan1,2,3()   

  1. 1. College of Pharmacy, Hunan University of Traditional Chinese Medicine, Changsha 410208
    2. Key Laboratory for the Germplasm Resources and Standardized Planting of Hunan's Bulk Authentic Medicinal Materials, Changsha 410208
    3. Hunan Provincial Key Laboratory of Traditional Chinese Medicine Modernization, Changsha 410208
  • Received:2023-03-06 Published:2023-10-26 Online:2023-11-28

摘要:

克隆灰毡毛忍冬糖基转移酶UGTPg17UGTPg36基因,并进行其不同花期表达量与皂苷含量相关性分析。根据灰毡毛忍冬转录组Unigene序列设计特异性引物进行UGTPg17UGTPg36克隆;使用生物信息学网站对克隆的UGTPg17、UGTPg36编码蛋白进行理化性质、蛋白结构和进化关系等分析;实时荧光定量PCR(RT-qPCR)分析基因在不同花期表达情况;HPLC法测定灰毡毛忍冬皂苷含量;利用SPSS分析基因表达量与皂苷含量相关性。UGTPg17、UGTPg36基因开放阅读框长度分别为1 410 bp、1 428 bp,分别编码469、475个氨基酸,分别包含4、7个糖基化位点,均属于不稳定、亲水性蛋白,不具跨膜区域;UGTPg36蛋白不存在信号肽序列,UGTPg17蛋白平均S值>0.5,推测其可能含有信号肽。UGTPg17UGTPg36在不同花期中表达均为上升趋势,皂苷含量在不同花期中存在波动,两者相关性分析得出,2个UGT基因与皂苷含量呈极显著负相关。成功从灰毡毛忍冬中克隆了UGTPg17UGTPg36基因,其在不同花期表达存在差异性,且相关性分析表明UGTPg17UGTPg36基因与灰毡毛忍冬皂苷生物合成相关,为进一步探索其具体功能奠定了基础。

关键词: 灰毡毛忍冬, 糖基转移酶, 基因克隆, 生物信息学, 表达分析

Abstract:

This work is to clone the genes of glycosyltransferases UGTPg17 and UGTPg36 from Lonicera macranthoides, and analyze the correlation between their expression and saponin content at different flowering stages. According to the Unigene sequence of L. macranthoides transcriptome, specific primers were designed to clone UGTPg17 and UGTPg36. The physical and chemical properties, protein structure and evolutionary relationship of the cloned UGTPg17- and UGTPg36-encoding proteins were analyzed using the bioinformatics websites; quantitative reverse-transcription polymerase chain reaction(RT-qPCR)was used to analyze gene expressions at different flowering stages. The content of L. macranthoides saponin was determined by HPLC; and the correlation between gene expression and saponin content was analyzed by SPSS. The open reading frame length of UGTPg17 and UGTPg36 genes was 1 410 bp and 1 428 bp, respectively, encoding 469 and 475 amino acids, containing 4 and 7 glycosylation sites, respectively, which belonged to unstable and hydrophilic proteins without transmembrane region. UGTPg36 protein did not contain signal peptide sequence, and the average S value of UGTPg17 protein was >0.5, suggesting that it may contain signal peptide. The expressions of UGTPg17 and UGTPg36 in different flowering stages showed an upward trend, and the saponin content fluctuated in different flowering stages. The correlation analysis between the two showed that the two UGT genes were significantly negatively correlated with the saponin content. In this study, UGTPg17 and UGTPg36 genes were successfully cloned from L. macranthoides, and their expressions variedt at different flowering stages. Correlation analysis showed that UGTPg17 and UGTPg36 genes were related to saponin biosynthesis of L. macranthoides, which lays a foundation for further exploring their specific functions.

Key words: Lonicera macranthoides, glycosyltransferase, gene cloning, bioinformatics, expression analysis