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    张星耀, 覃庆锋, 贺伟, 梁军, 刘会香. 毛白杨巯基蛋白酶抑制剂基因cDNA的克隆及序列分析[J]. 北京林业大学学报, 2007, 29(6): 23-28. DOI: 10.13332/j.1000-1522.2007.06.010
    引用本文: 张星耀, 覃庆锋, 贺伟, 梁军, 刘会香. 毛白杨巯基蛋白酶抑制剂基因cDNA的克隆及序列分析[J]. 北京林业大学学报, 2007, 29(6): 23-28. DOI: 10.13332/j.1000-1522.2007.06.010
    ZHANG Xing-yao, TAN Qing-feng, HE Wei, LIANG Jun, LIU Hui-xiang. Cloning and sequence analysis on cDNA of cysteine proteinase inhibitor gene from Populus tomentosa Carr[J]. Journal of Beijing Forestry University, 2007, 29(6): 23-28. DOI: 10.13332/j.1000-1522.2007.06.010
    Citation: ZHANG Xing-yao, TAN Qing-feng, HE Wei, LIANG Jun, LIU Hui-xiang. Cloning and sequence analysis on cDNA of cysteine proteinase inhibitor gene from Populus tomentosa Carr[J]. Journal of Beijing Forestry University, 2007, 29(6): 23-28. DOI: 10.13332/j.1000-1522.2007.06.010

    毛白杨巯基蛋白酶抑制剂基因cDNA的克隆及序列分析

    Cloning and sequence analysis on cDNA of cysteine proteinase inhibitor gene from Populus tomentosa Carr

    • 摘要: 为了发掘我国乡土树种毛白杨巯基蛋白酶抑制剂(CPI)基因资源,该研究在对已知植物巯基蛋白酶抑制剂氨基酸序列保守性和杨树表达序列标签(EST)序列分析的基础上,设计了1对简并引物和1对杨树特异性CPI引物,应用RT-PCR技术在毛白杨形成层中扩增出了一个696 bp的cDNA片段,将此片段连接到pGEM-T Easy载体上并测序.结果表明,该片段含有一个417 bp的开放阅读框,编码138个氨基酸残基.进一步对氨基酸序列分析表明,该氨基酸具有典型植物巯基蛋白酶抑制剂的保守区段,即靠近氨基端具有甘氨酸(G)残基和LVI-AGT-RKE-FY-AS-VI-x-EDQV-HYFQ序列,羧基端具有与酶活性有关的QXVXG结构和PW残基,说明毛白杨形成层中存在CPI基因并能够有效表达.氨基酸序列同源性分析表明,该基因与其他林木CPI的同源性在47%~68%之间.

       

      Abstract: Plant cysteine proteinase inhibitors(CPI,phytocystatins) have been widely used in the plant pest-resisting genetic engineers.The aim of this study is to isolate the CPI gene from an indigenous tree,Populus tomentosa Carr.Based on the analysis of the conserved amino acid sequences of reported CPI and poplar expressed sequence tags(EST),two pairs of degenerate and poplar-specific primers were designed for reverse transcription polymerase chain reaction(RT-PCR).A fragment containing 696 base pairs(bp) of nucleotide was amplified from total cDNA of P.tomentosa cambium.Sequencing was carried out through linking to pGEM-T Easy vector.Sequence analysis showed that the cDNA fragment included an open reading frame(ORF) of 417 bp in length,which encoded 138 amino acid residues.Deep analysis of amino acid sequences shows that it contains four conserved motifs region of phytocystatins,which are G residue near the N-terminus and LVI-AGT-RKE-FY-AS-VI-x-EDQV-HYFQ,active site motif QxVxG and PW residues at the C-terminus,these results prove that the CPI gene exists and is effectively expressed in P.tomentosa cambium.Homology analysis of CPI amino acid sequences resulted in 47%-68% similarity scores between P.tomentosa and other trees.

       

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