高级检索
    李兴芬, 苗雅慧, 孙永江, 张孟娟, 张凌云. 青杄PwPEBP基因及其启动子序列的克隆与表达分析[J]. 北京林业大学学报, 2019, 41(4): 8-20. DOI: 10.13332/j.1000-1522.20180344
    引用本文: 李兴芬, 苗雅慧, 孙永江, 张孟娟, 张凌云. 青杄PwPEBP基因及其启动子序列的克隆与表达分析[J]. 北京林业大学学报, 2019, 41(4): 8-20. DOI: 10.13332/j.1000-1522.20180344
    Li Xingfen, Miao Yahui, Sun Yongjiang, Zhang Mengjuan, Zhang Lingyun. Cloning and expression analysis of PwPEBP gene and promoter sequence in Picea wilsonii[J]. Journal of Beijing Forestry University, 2019, 41(4): 8-20. DOI: 10.13332/j.1000-1522.20180344
    Citation: Li Xingfen, Miao Yahui, Sun Yongjiang, Zhang Mengjuan, Zhang Lingyun. Cloning and expression analysis of PwPEBP gene and promoter sequence in Picea wilsonii[J]. Journal of Beijing Forestry University, 2019, 41(4): 8-20. DOI: 10.13332/j.1000-1522.20180344

    青杄PwPEBP基因及其启动子序列的克隆与表达分析

    Cloning and expression analysis of PwPEBP gene and promoter sequence in Picea wilsonii

    • 摘要:
      目的通过研究青杄中的PwPEBP基因及其启动子的表达特性及生物学功能,探究PEBP基因在植物生长发育过程中响应逆境胁迫的功能及作用机制。
      方法本研究从青杄转录组测序中获得PwPEBP的cDNA序列,利用TMHMM、GOR4等在线软件对PwPEBP蛋白进行生物信息学分析,以此为基础通过PCR技术克隆得到PwPEBP开放阅读框(ORF)序列;同时对其在不同组织与不同逆境及激素处理中的表达水平进行了RT-qPCR技术分析;采用染色体步移法克隆PwPEBP的启动子序列,并利用在线软件BDGP和PlantCARE对PwPEBP启动子序列进行基础启动子区域、转录起始位点和顺式作用元件的预测;最后通过农杆菌瞬时转化烟草法验证PwPEBP启动子的功能。
      结果PwPEBP cDNA长度为1 408 bp,开放阅读框共585 bp,编码194个氨基酸。PwPEBP蛋白分子式为C966H1 484N250O299S6,无信号肽和跨膜结构域,为亲水蛋白,含有25个磷酸化位点;进化树分析显示,PwPEBP蛋白与北美云杉的PEBP单独聚成一簇,属于新的PEBP蛋白。组织特异性分析显示,PwPEBP在成熟叶中表达量最高,嫩叶中表达量最低;PwPEBP在各激素及逆境诱导下均有表达,但对盐处理无响应。克隆的PwPEBP启动子序列长度为903 bp,其具有响应GA、ABA、SA、MeJA的顺式作用元件。GUS染色及Luc定量实验显示,PwPEBP启动子均能响应GA、ABA、MeJA和SA外源激素及干旱、高温、低温等逆境胁迫。
      结论青杄中PwPEBP基因广泛响应干旱、低温、高温等非生物胁迫,其中对干旱胁迫最为敏感,同时还参与了ABA、GA、MeJA和SA激素的信号通路。

       

      Abstract:
      ObjectiveThrough the research of PwPEBP gene and its promoter expression characteristic and biological function in Picea wilsonii, this paper aims to explore PEBP genes in plant growth and development in the process of response to adversity stress function and mechanism of action.
      MethodIn this study, we had obtained the cDNA sequence of PwPEBP by Picea wilsonii transcriptome data. Based on the bioinformatics analysis of PwPEBP protein by TMHMM, GOR4 and other online software, the open reading frame (ORF) sequence of PwPEBP was cloned by PCR technology. The expression level of the gene in different tissues, different adversities and hormone treatments was analyzed by RT-qPCR. The promoter sequence of PwPEBP was cloned by chromosome step method, and the prediction of the basic promoter region, transcription starting point and the action element of the PwPEBP promoter sequence was carried out by the on-line software BDGP and PlantCARE. Finally, the function of PwPEBP promoter was transferred into tobacco leaves by agrobacterium-mediated method.
      ResultPwPEBP cDNA was 1 408 bp with an open reading frame flanked (ORF) of 585 bp encoding 194 amino acids.The protein molecular formula of PwPEBP was C966H1 484N250O299S6. The protein had no peptide and transmembrane domain. Hydrophobicity analysis showed that the hydrophobic sites of PwPEBP were uniformly distributed, suggesting that the protein was hydrophilic.25 phosphorylation sites were found with NetPhos. The phylogenetic tree analysis showed that the PwPEBP and PEBP of the Picea sitchensis were clustered into a new PEBP protein. The tissue-specific expression analysis showed that PwPEBP was all expressed in stem, root, mature needle, young needle, pollen and seed. However, the expression level of PwPEBP in mature needle was the highest and the lowest in young needle. The expression of PwPEBP under stress and hormone treatments showed that the expression of PwPEBP was induced, respectively, but not by the salt stress.The sequence length of PwPEBP promoter was 903 bp. The online analysis showed that it contained cis-acting elements such as GA, ABA, MeJA and SA.The GUS color reaction and Luc quantification experiment further showed that PwPEBP promoter sequences with cis elements could respond to GA, ABA, MeJA and SA hormones.
      ConclusionPwPEBP gene in Picea wilsonii widely responds to abiotic stress such as drought, low temperature and high temperature, especially in drought. Meanwhile, PwPEBP is also involved in the signaling pathways of ABA, GA, MeJA and SA hormones.

       

    /

    返回文章
    返回