地钱MpPP2A-A基因克隆及基因敲除突变体构建

MpPP2A-A gene cloning and knockout mutant constructing in Marchantia polymorpha

关键词:  PP2A, 地钱, 基因克隆, CRISPR/Cas9, 载体构建

Key words:  PP2A, Marchantia polymorpha, gene cloning, CRISPR/Cas9, vector construction

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。

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.