生物技术通报 ›› 2022, Vol. 38 ›› Issue (9): 198-206.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0510

• 研究报告 • 上一篇    下一篇

马缨杜鹃Rd3GT1的克隆及对矮牵牛花色形成的影响

孙威1(), 张艳1, 王聿晗1, 徐僡1, 徐小蓉1(), 鞠志刚2()   

  1. 1.贵州师范大学生命科学学院 植物生理与发育调控重点实验室, 贵阳 550025
    2.贵州中医药大学药学院,贵阳 550025
  • 收稿日期:2022-04-25 出版日期:2022-09-26 发布日期:2022-10-11
  • 作者简介:孙 威,女,博士,副教授,研究方向:植物生物技术;E-mail: sunwei889@163.com
  • 基金资助:
    国家自然科学基金项目(31760076);贵州省教育厅特色领域项目(黔教合KY字[2021]059);贵州省科学技厅项目(黔科合支撑[2020]4Y028号);贵州省科学技厅项目(黔科合基础[2019]1019)

Cloning of Rd3GT1 in Rhododendron delavayi and Its Effect on Flower Color Formation of Petunia hybrida

SUN Wei1(), ZHANG Yan1, WANG Yu-han1, XU Hui1, XU Xiao-rong1(), JU Zhi-gang2()   

  1. 1. Key Laboratory of Plant Physiology and Development Regulation, School of Life Science, Guizhou Normal University, Guiyang 550025
    2. Pharmacy College, Guizhou University of Traditional Chinese Medicine, Guiyang 550025
  • Received:2022-04-25 Published:2022-09-26 Online:2022-10-11

摘要:

类黄酮3-O-糖基转移酶(3GT)是花色素苷生物合成途径末端的酶,负责将糖基供体转移至花青素的3-OH位置,在增加花色素苷的稳定性与水溶性方面发挥重要作用。研究马缨杜鹃3GT在花色素苷生物合成中的功能,为马缨杜鹃花色形成及调控研究奠定基础。运用 RT-PCR 技术克隆获得马缨杜鹃3GT基因(Rd3GT1)全长CDS序列,并对其进行生物信息学分析,再利用DNA重组技术完成植物表达载体pBI121-Rd3GT1的构建,利用农杆菌介导法对矮牵牛进行遗传转化,同时对再生植株进行转基因及表型鉴定,并完成基因表达量及花色素苷检测分析。结果表明,Rd3GT1全长CDS序列为1 395 bp,编码464个氨基酸。蛋白多序列比对和系统进化分析表明,Rd3GT1属于3GT家族成员。与野生型相比,转Rd3GT1的矮牵牛植株花色由白色变为粉色,花色素苷与黄酮醇的积累显著增加,同时多个类黄酮合成相关基因表达显著升高。综上,Rd3GT1在花色素苷合成过程中发挥重要功能,可用于其它植物花色改良研究。

关键词: 马缨杜鹃, 克隆, Rd3GT1, 遗传转化, 花色素苷

Abstract:

Flavonoid 3-O-glycosyltransferase(3GT)is an enzyme at the end of anthocyanin biosynthetic pathway, responsible for transferring glycosylated donors to the 3-OH site of anthocyanidin, and plays an important role in increasing the stability and water-solubility of anthocyanin. To study the function of 3GT in Rhododendron delavayi during anthocyanin biosynthesis would provide the basis for the research on R. delavayi flower color formation and its regulation. The full length CDS of Rd3GT1 was cloned by RT-PCR and analyzed by bioinformatics, then the pBI121-Rd3GT1 plant expression vector was constructed by DNA recombination technology. Subsequently, petunia hybrida was transformed by Agrobacterium-mediated method,and the regenerated plants were identified by transgene detection, phenotype identification, gene expression and anthocyanin detection analysis. The results showed that the full length CDS of Rd3GT1 was 1 395 bp encoding 464 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that Rd3GT1 belonged to 3GT subfamily. Compared with the wild type, the flower color of Rd3GT1 transgenic petunias changed from white to pink, anthocyanin and flavonol accumulations as well as expressions of several flavonoid-related biosynthetic genes significantly increased. In conclusion, Rd3GT1 plays a vital role in anthocyanin synthesis and can be used to improve flower color in other plants.

Key words: Rhododendron delavayi, cloning, Rd3GT1, genetic transformation, anthocyanin