TANG Caiqun,LEI Xiujin,ZHAO Yan,et al.Cloning，Expression，and Functional Analysis of the TsMYB1 Gene，Encoding a R2R3-MYB Transcription Factor From Toona sinensis[J].Northern Horticulture,2025,(13):8-17.[doi:doi：10.11937/bfyy.20244410]
香椿R2R3-MYB转录因子基因TsMYB1克隆、表达与功能分析
- Title:
- Cloning，Expression，and Functional Analysis of the TsMYB1 Gene，Encoding a R2R3-MYB Transcription Factor From Toona sinensis
- 文章编号:
- 1001-0009(2025)13-0008-10
- 关键词:
- 香椿; R2R3-MYB转录因子; 花青素
- Keywords:
- Toona sinensis; R2R3-MYB transcription factor; anthocyanin
- 分类号:
- S 644.4
- 文献标志码:
- A
- 摘要:
- 以‘红香椿’嫩叶为试材，采用RT-PCR技术克隆了R2R3-MYB转录因子基因TsMYB1，利用生物信息学方法分析该基因的序列结构、保守结构域、进化关系和蛋白质三维结构、理化性质，采用RT-qPCR技术分析该基因组织特异性表达模式，构建过表达载体，利用农杆菌介导的浸花法转化拟南芥，验证功能，以期为深入解析该基因在香椿花青素合成中的功能提供参考依据。结果表明：TsMYB1基因开放阅读框为831 bp，编码1个由276个氨基酸组成、相对分子量为31.427 kDa、理论等电点为8.24的稳定亲水碱性蛋白。TsMYB1蛋白含有R2R3-MYB蛋白家族保守的R2和R3结构域。二级结构预测其α-螺旋、β-转角、β-折叠和无规则卷曲所占比例分别为28.99%、5.43%、9.42%和56.16%。该基因编码的氨基酸序列与MaMYB序列相似度达69.93%，进化关系较为密切。TsMYB1在香椿各组织中有特异性表达，在叶中表达量最高，其次是茎，在根中表达量最低。过表达TsMYB1的拟南芥植株叶片花青素积累增加，叶色由绿变紫。TsMYB1基因编码1个R2R3-MYB转录因子，其表达具有组织特异性，具有促进植物花青素合成功能。
- Abstract:
- Taking the tender leaf of ‘Red Toona sinensis’ as experimental materials，the full-length cDNA sequence of R2R3-MYB transcription factor gene TsMYB1 was cloned by using RT-PCR technique，and using bioinformatics methods to analyzed sequence structure，conserved domain，evolutionary relationships，protein three-dimensional structure，and the physicochemical properties.The special expression patterns in different tissues were studied by real-time quantitative RT-PCR (RT-qPCR)，TsMYB1 over expression vector was constructed and then was transformed into wild-type Arabidopsis thaliana by Agrobacterium-mediated floral dip method to verify its function，in order to provide reference for further analysis of the function of TsMYB1 in anthocyanin biosynthesis in Toona sinensi.The results showed that TsMYB1 had an open reading frame of 831 bp and encodes a stable alkaline hydrophilic protein consisting of 276 amino acids with a relative molecular mass of 31.427 kDa and a theoretical isoelectric point of 8.24.The TsMYB1 protein contained the conserved R2 and R3 conserved structural domains of the R2R3-MYB family.The predicted secondary structure contained α-helix，β-turn，β-sheet，and random coil，which had 28.99%，5.43%，9.42%，and 56.16%，respectively.Phylogenetic analysis revealed that TsMYB1 had a close genetic relationship of 69.93% similarity with Melia azedarach homologous gene MaMYB.RT-qPCR confirmed that TsMYB1 in the leaves，followed by the stem，and low expression in roots.Overexpression of TsMYB1 promoted the synthesis of anthocyanin in Arabidopsis，and resulted in a change in leaves′ color from green to purple.This study clarified that TsMYB1 encoded an R2R3-MYB transcription factor，showed tissue-specific expression patterns and promoted anthocyanin synthesis in plants.
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备注/Memo
第一作者简介：唐彩群（1999-），女，硕士研究生，研究方向为植物次生代谢产物生物学功能。E-mail：20221002094@stu.ymun.edu.cn.责任作者：徐启江（1969-），男，博士，教授，现主要从事被子植物分子系统发育遗传学、药用植物功能小分子合成机制与药理学及植物分子育种等研究工作。E-mail：qijiangxu@126.com.基金项目：2022与2023年百色市科学研究与技术开发计划资助项目（百科2022002，百科20230533，百科20230534，百科20230535）。收稿日期：2024-11-08