平邑甜茶新根扩展蛋白基因MhEXP1的cDNA全序列克隆及表达

Cloning and Expression of a Full-length cDNA of Expansin Gene from New Root of Malus hupehensis Rehd.

【目的】对平邑甜茶[Malus hupehensis (Pamp) Rehd.var pinyiensis Jiang]新根扩展蛋白基因进行克隆和表达分析,为揭示扩展蛋白在平邑甜茶根系生长发育中的功能打下基础。【方法】利用RT-PCR结合RACE技术克隆扩展蛋白同源基因,并通过Northern杂交研究其在不同器官中的表达情况及吲哚丁酸(IBA)的处理效应。【结果】成功地从平邑甜茶白色新根中获得了一个全长的扩展蛋白基因,命名为MhEXP1,GenBank登录号为DQ538346。MhEXP1 cDNA全长1111bp,含有771bp的完整开放阅读框,编码257个氨基酸,预测分子量和等电点分别为27.8kD和8.9。序列分析表明,MhEXP1具有扩展蛋白的典型结构,即氨基酸序列N端有8个半胱氨酸残基,1个组氨酸(His-Phe-Asp,HFD)域,C末端存在4个保守的色氨酸残基。Northern杂交表明,该基因在平邑甜茶叶片中表达量很低,但在新根中大量表达并受IBA诱导,且随着IBA处理时间的延长表达量逐渐增加。【结论】成功地从平邑甜茶中克隆了一个全长的扩展蛋白基因MhEXP1,该基因在叶片中表达量很低但在新根中大量表达,并受IBA诱导;MhEXP1参与IBA调节的平邑甜茶根系生长发育。

[Objective] The aim of this research is to clone and investigate the expression of the expansin gene from new root of Malus hupehensis （Pamp） Rehd. vat pinyiensis Jiang, which will be helpful to study the action of expansin gene in root development. [ Method ] With the RT-PCR and RACE methods, a full length cDNA sequence of expansin gene was cloned in root of Malus hupehensis （Pamp） Rehd. The expression of expansin gene was analyzed by Northern blot. [Result] Sequencing and structural analysis showed that the full-length of MhEXP1 consisted of I 111 bp with an open reading frame of 771 bp that could code for a protein of 257 amino acids. The predicted MW and PI were 27.8 kD and 8.9. The polypeptide had a N-terminal signal peptide of 23 amino acids like many other expansins, Essential features of the protein such as the eight cysteine residues and four tryptophan residues near the C-terminal end are conserved in the sequence, The HFD sequence, which has been shown to be a part of the endoglucanase active site is also present in MhExpl. Northern blot showed that the expression ofMhEXP1 gene was much higher in roots than that in leaves. The transcriptional level of the MhEXP1 gene in the roots of Malus hupehensis （Pamp） Rehd increased by 10 μmol/L IBA treatment for 6 h. [ Conclusion ] All of the results indicated that the obtained gene of MhEXP1 is a new member of expansin gene family. The expression of MhEXP1 gene is much higher in roots than that in leaves. The transcription of MhEXP1 is regulated by IBA and it plays an important role in root development of Malus hupehensis （Pamp） Rehd.