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    黄冠梨PbpNCED3基因的克隆与表达

    Cloning and expression of PbpNCED3 gene from ‘Huangguan’ pear

      摘要
    • 摘要:
      目的  克隆黄冠梨Pyrus bretschneideri‘Xuehuali’ × P. pyrifolia ‘Shinsseiki’叶片中的9−顺式−环氧类胡萝卜素裂解双加氧酶(NCED)基因的全长cDNA序列，研究不同程度干旱条件下该基因的表达及与叶片内源脱落酸(ABA)的动态变化关系，为明晰该基因在梨树抗旱中的分子机理提供理论依据。
      方法  根据转录组数据得到的PbpNCED3序列进行同源序列扩增，通过生物信息学分析确定其属于NCED家族基因。通过酶联免疫吸附法测定不同水分条件下梨叶片内源ABA的含量，通过实时荧光定量测定PbpNCED3基因的表达量。
      结果  PbpNCED3cDNA序列全长为1 831bp，编码603个氨基酸(GeneBank登录号为：MH749461)。该蛋白具有NCED发挥功能所依赖的4个保守的组氨酸，有NCED家族特有保守结构域序列MIAHPKxDP和HDFAITE及RPE65结构域，N−端含有靶向叶绿体的转运肽。蛋白结构预测发现，该蛋白与玉米vp14(NCED家族蛋白)比对序列相似性为70%，且具有两亲性的α螺旋结构。PbpNCED3基因的表达量与梨叶片ABA的含量成正相关关系(相关系数为0.914)，且二者都受到干旱的诱导。
      结论  试验所得到的黄冠梨PbpNCED3属于NCED家族基因，该基因响应干旱胁迫，其表达量与内源ABA的含量动态变化趋势一致。

       

      Abstract:
      Objective  To clone the full-length cDNA sequence of 9-cis-epoxycarotenoid dioxygenase (NCED) gene from leaves of Pyrus bretschneideri‘Xuehuali’ × P. pyrifolia ‘Shinsseiki’ cultivar ‘Huangguan’, study the relationship between its expression and the endogenous abscisic acid (ABA) content in leaves under different degree of drought, and provide a theoretical basis for understanding the molecular mechanism of the gene in drought resistance of pear tree.
      Method  According to the PbpNCED3 sequence obtained from the transcriptome data, the full-length cDNA sequence was cloned by homologous sequence amplification. The sequence was identified as belonging to the NCED family by bioinformatics analysis. Endogenous ABA content in pear leaves under different soil moisture conditions was determined by enzyme-linked immunosorbent assay (icELISA), and the expression of PbpNCED3 gene was measured by real time qPCR.
      Result  The PbpNCED3 cDNA sequence is 1 831 bp encoding 603 amino acids. The protein has four conserved histidines required for activity, two conservative domain (MIAHPKxDP and HDFAITE) characteristic of the NCED family and a RPE65 domain. There is a putative chloroplast-targeting peptide at N-terminal. The protein structure prediction shows that the protein sequence has 70% similarity to vp14 (NCED family protein) and it has a amphipathic α helix structure. PbpNCED3 gene expression is positively related with ABA content in pear leaves with a correlation coefficient of 0.914, and both of them are induced by drought.
      Conclusion  The PbpNCED3 gene obtained from the experiment belongs to the NCEDfamily. It responses to drought stress and its expression shares a similar dynamic trend with ABA content in pear leaves.

       

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