| 引用本文: | 刘文珍, 江昕桦, 张邦跃, 陈 莎, 张晶晶, 李相媛, 荣朵艳.地钱MpPP2A-A基因克隆及基因敲除突变体构建[J].广西植物,2025,45(7):1323-1335.[点击复制] |
| LIU Wenzhen, JIANG Xinhua, ZHANG Bangyue, CHEN Sha,
ZHANG Jingjing, LI Xiangyuan, RONG Duoyan.MpPP2A-A gene cloning and knockout mutant constructing in Marchantia polymorpha[J].Guihaia,2025,45(7):1323-1335.[点击复制] |
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| 地钱MpPP2A-A基因克隆及基因敲除突变体构建 |
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刘文珍1,2,3, 江昕桦1,2, 张邦跃1,2, 陈 莎1,2, 张晶晶1,2, 李相媛1,2, 荣朵艳1,2,3*
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1. 湖南工业大学 生物与医学工程学院, 湖南 株洲 412007;2. 百合种质资源创新与深加工湖南省工程研究中心,
湖南 株洲 412007;3. 株洲市环境微生物与植物资源利用联合实验室, 湖南 株洲 412007
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| 摘要: |
| 蛋白磷酸酶2A(protein phosphatase 2A,PP2A)是一种丝氨酸/苏氨酸磷酸酶,通过去磷酸化底物蛋白参与植物的生长发育等生物学过程。地钱(Marchantia polymorpha)是一种新兴的模式植物,具有基因组小且基因冗余性低等许多优势。为了探究PP2A在植物生长中的调控机制,该文以地钱为研究对象,克隆了MpPP2A-A亚基编码区全长,并利用生物信息学软件和实时荧光定量PCR技术对MpPP2A-A基因的组织表达情况进行了分析,同时构建了MpPP2A-A基因的敲除突变体。结果表明:(1)MpPP2A-A基因编码区全长1 761 bp,编码586个氨基酸,含有3个结构域,不具有信号肽。(2)氨基酸序列比对结果显示,PP2A-A在植物进化过程中相对保守。(3)实时荧光定量PCR结果显示,MpPP2A-A基因在顶端缺口处、叶状体、胞芽杯中的表达依次减弱。(4)通过CRISPR/Cas9技术成功获得了3个独立的突变体株系,统计发现突变体胞芽面积较野生型Tak1显著性减小且其形态异常。该研究结果表明MpPP2A-A基因在地钱胞芽的生长过程中发挥着重要作用,为今后进一步探究其调控植物生长发育的分子机制奠定了基础。 |
| 关键词: PP2A, 地钱, 基因克隆, CRISPR/Cas9, 载体构建 |
| DOI：10.11931/guihaia.gxzw202406014 |
| 分类号:Q943.2 |
| 文章编号:1000-3142(2025)07-1323-13 |
| 基金项目:国家自然科学基金青年项目(32100576); 湖南省自然科学基金优秀青年项目(2024JJ4018); 湖南省教育厅优秀青年基金项目(23B0541)。 |
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| MpPP2A-A gene cloning and knockout mutant constructing in Marchantia polymorpha |
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LIU Wenzhen1,2,3, JIANG Xinhua1,2, ZHANG Bangyue1,2, CHEN Sha1,2,
ZHANG Jingjing1,2, LI Xiangyuan1,2, RONG Duoyan1,2,3*
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1. School of Biological Science and Medical Engineering, Hunan University of Technology, Zhuzhou 412007, Hunan, China;2. Hunan
Engineering Research Center for Lilium Germplasm Innovation and Deep Processing, Zhuzhou 412007, Hunan, China;3. Zhuzhou
City Joint Laboratory for Environmental Microbiology and Plant Resource Utilization, Zhuzhou 412007, Hunan, China
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| Abstract: |
| Protein phosphatase 2A(PP2A), a serine/threonine phosphatases, participates in many biological processes such as plant growth and development by dephosphorylating its substrate proteins. Marchantia polymorpha is an emerging model plant with many advantages such as a small genome and low gene redundancy. To explore the regulatory mechanism of PP2A in plant growth, the full-length coding region of MpPP2A-A subunit was cloned from M. polymorpha, and its expression was analyzed by using bioinformatics software and real-time fluorescent quantitative PCR technology. At the same time, a knockout mutant of MpPP2A-A gene was constructed. The results were as follows:(1)The full-length coding region of the MpPP2A-A gene was 1 761 bp, encoding 586 amino acids, containing three domains and no signal peptide.(2)The amino acid sequence alignment analysis indicated that PP2A-A was relatively conserved during plant evolution.(3)Real-time fluorescent quantitative PCR showed that the expression of MpPP2A-A gene in the apical notch, thallus, and gemma cup decreased successively.(4)Three independent mutant lines were successfully obtained by CRISPR/Cas9 technology. It was found that the area of the mutant gemma was significantly reduced compared with wild-type Tak1, and its morphology was abnormal. The research results show that MpPP2A-A gene plays an important role in the growth process of M. polymorpha gemma, laying a foundation for further exploring the molecular mechanism of PP2A-A gene in regulating plant growth and development in the future. |
| Key words: PP2A, Marchantia polymorpha, gene cloning, CRISPR/Cas9, vector construction |
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