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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。
The cDNA library of SSH was constructed by screening Red Suppression Buds of Malus domestica CV Red Fuji and its stripe rust parental strain, and screening SSH-cDNA library by differential screening. Fortune Red Fuji apple EST sequence of local databases, the local database Blastn search to obtain 42 pieces of metallothionein gene MdFjMT2 cDNA fragments obtained by sequence stitching and reverse transcription PCR (RT-PCR) method, Fuji apple metallothionein The full-length cDNA of MdFjMT2 was 684bp, including 97bp in 5 ’untranslated region, 347bp in 3’ untranslated region, 240bp in coding region (CDS) and 79 in GenBank accession number HQ730757 Amino acids, suggesting that the protein molecular weight of 7.7938kDa, the theoretical isoelectric point of 4.75. This gene has the typical domain characteristic of metallothionein gene. The encoded amino acid sequence contains 14 cysteine residues, Cys (C). The arrangement of Cys residues is characterized by the concentration of CC, CXC and CXXC in the peptide chain N-terminal and C-terminal. Phylogenetic analysis showed that MdFjMT2 had a close genetic relationship with plant metallothioneins such as Pyrus pyrifo-lia, Malus domestica and M. xiaojinensis, and was closely related to Prunus dulcis, Salixmatsudana, Actinidia deliciosa and other plants. Bioinformatics analysis showed that MdFjMT2 is mainly located in the chloroplast and has no signal peptide. It is a non-transmembrane hydrophilic protein. The main component of secondary structure of MdFjMT2 is random curl and no functional domain. These results provide a reference for the structure and function mining of Md-FjMT2. The results of this study are helpful to study the role of this gene in apple coloring and elucidate the molecular mechanism of apple coloring.