牡丹PsMADS4基因的克隆、 表达分析及载体构建

姜纯,周陶敏,盖树鹏,张玉喜,刘春英*
青岛农业大学生命科学学院, 山东省高校植物生物技术重点实验室, 山东青岛266109

通信作者:刘春英;E-mail: ycliu2@163.com

摘 要:

以牡丹(Paeonia suffruticosa)花瓣为材料, 根据课题组前期得到的一条花衰老相关基因的表达序列标签, 通过RACE-PCR技术成功获得MADS-box转录因子家族中的PsMADS4基因的全长cDNA序列, 并对其进行了生物信息学分析。采用实时定量PCR技术对PsMADS4基因进行了时空表达分析, 构建了PsMADS4超表达载体。结果表明, PsMADS4cDNA全长1 081 bp, 开放阅读框长738 bp, 编码245个氨基酸;该蛋白的分子量为28.23 kDa, 等电点为8.513, 为疏水性蛋白; 表达分析结果显示, PsMADS4在不同组织中的表达水平不同, 在花瓣中的表达量最高, 且在盛花期花瓣中的表达量较高; pBI121-PsMADS4重组质粒分别进行PCR鉴定和双酶切鉴定, 结果显示PsMADS4基因已经插入植物表达载体pBI121, 表明PsMADS4的过表达载体构建成功。研究结果拟对进一步研究PsMADS4在牡丹花发育中的功能提供理论依据, 为延缓牡丹花衰老提供候选基因。

关键词:牡丹; 衰老; 克隆; 表达分析; 载体构建

收稿:2020-07-31   修定:2020-11-03

资助:国家自然科学基金(32072614、 31972452和31872145)、青岛农业大学高层次人才科研基金(6631116009)和山东省大学生创新创业 训练计划项目(S201910435045)

Cloning, expression analysis and vector construction of tree peony PsMADS4 gene

JIANG Chun, ZHOU Taomin, GAI Shupeng, ZHANG Yuxi, LIU Chunying*
Key Lab of Plant Biotechnology in Universities of Shandong Province, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: LIU Chunying; E-mail: ycliu2@163.com

Abstract:

The full-length cDNA sequence of PsMADS4 gene, which was a senescence-related gene, had been obtained from tree peony by RACE-PCR technology based on the expressed sequence tag (EST) of PsMADS4. The bioinformatics analysis was carried out and the spatiotemporal expression were analyzed

by real-time quantitative PCR. In additional, the overexpression vector of PsMADS4 gene was constructed. The results showed that PsMADS4 cDNA was 1 081 bp, and its open reading frame was 738 bp, which encoded a protein with 245 amino acids. The PsMADS4 protein was a hydrophobic protein, and its molecular weight and isoelectric point were 28.23 kDa and 8.513, respectively. Real-time quantitative PCR analysis revealed that the expression levels of PsMADS4 gene varied in different tissues, and the expression level was the highest in petals. It was worth mentioning that the expression level of PsMADS4 in petals was much higher in D3 stage than that in D6 stage. The results of PCR and double enzyme digestion indicated that PsMADS4 gene had been inserted into plant expression vector PBI121, and the overexpression vector of PsMADS4 gene had been constructed successfully. Our results would provide theoretical basis for further study on the function of PsMADS4 gene during the flower development, and offer candidate gene for delaying the flower senescence in tree peony.


Key words: tree peony; senescence; cloning; expression analysis; vector construction

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