苹果金属硫蛋白基因MdFjMT2克隆及生物信息学分析

Cloning and Bioinformatic Analysis of Metallothionein-like Protein Gene (MdFjMT2) from Apple(Malus domestica)

以红富士苹果(Malus domesticaCV Red Fuji)浓红型芽变和其条红母株为试材,构建抑制性差减杂交(Suppression Subtractive Hybridization,SSH)文库,差异筛选SSH-cDNA文库,经过大量测序构建成红富士苹果EST序列本地数据库,对本地数据库进行Blastn检索,得到42条金属硫蛋白基因MdFjMT2的cDNA片段,通过序列拼接和逆转录PCR(RT-PCR)方法,获得了红富士苹果金属硫蛋白基因MdFjMT2全长cDNA序列(Gen-bank登录号为HQ730757),该基因全长684bp,其中5′非翻译区97bp,3′非翻译区347bp,编码区(Coding sequence,CDS)为240bp,编码79个氨基酸,推测蛋白分子量为7.7938kDa,理论等电点为4.75。该基因具有金属硫蛋白基因的典型结构域特征,编码的氨基酸序列中含有14个半胱氨酸残基Cys(C),Cys残基的排列特征是以CC、CXC和CXXC集中分布在肽链的N端和C端。系统进化分析表明MdFjMT2与砂梨(Pyrus pyrifo-lia)、苹果(Malus domestica)和小金海棠(M.xiaojinensis)等植物金属硫蛋白具有较近的亲缘关系,与扁桃(Prunus dulcis)、旱柳(Salixmatsudana)、美味猕猴桃(Actinidia deliciosa)等植物的亲缘关系较远。生物信息学分析结果表明,MdFjMT2主要位于叶绿体中,没有信号肽,是非跨膜亲水性蛋白,其蛋白质二级结构的主要元件是无规则卷曲,没有功能结构域。这些结果为Md-FjMT2的结构与功能挖掘提供一定的参考。本研究结果有助于研究该基因在苹果着色中的作用,阐明苹果着色的分子机制。

An apple EST local database of apple fruit （Malus domestica cv Red Fuji） was constructed by differential screening and a large number of sequencing against the suppression subtractive hybridization cDNA library from M. domestica cuhivar Red Fuji coloring type sports and its wild type. A metallothionein （MT） gene. named MdFjMT2 （Accession No. HQ730757） was cloned by RT-PCR and splicing of 42 cDNA sequences that were obtained by Blastn searching against the EST local database. The full length cDNA encoding metallothionein gene MdFjMT2 was 684bp. including 97bp of 5＇ untranslated region and 347bp of 3＇ untranslated region. It had a coding sequence （CDS） of 240bp. encoding 79 amino acid residues. The protein molecular weight was 7. 7938 kD and the theoretical pI was 4.75. Gene MdFjMT2 contained the conserved domains of all known MT. with 14 Cys （C） residues. ranking in the feature of CC,CXC,and CXXC, and locating at protein N terminus and C terminus. The phylogenetic tree analysis revealed that the amino acids sequence of MdFjMT2 shared higher similarity with Pyrus Pyrifo- lia M. domestica, and M. xiaofinensis, but lower similarity with Prunus dulcis, Salix matsudana, and Actinidia delicio-sa. Bioinformatio analysis showed that MdFjMT2 was mainly located in chloroplasts. It had no signal peptide and belonged to the hydrophilic non-transmembrane protein. MdFjMT2 had no functional domains and random coil was the major components of its secondary structure. These results provided information for further structure and functional study of MdFjMT2. The results would be useful to study the functions of gene,and clarify molecular mechanisms of apple coloring.