Cloning, expression analysis, and subcellular position of MdPH1 related to acidity in Malus domestica Borkh
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摘要: 以苹果属（Malus）植物沧江海棠（M.ombrophila Hand.-Mazz）的果实为材料，对其发育过程中苹果酸的含量进行测定，并结合转录组测序的方法筛选控制果实酸度的候选基因。结果显示：MdPH1候选基因的编码区包含2829 bp，编码942个氨基酸；基因组序列全长为4269 bp，包含8个外显子和7个内含子。对10份苹果种质资源中PH1基因序列的分析结果表明，该基因序列中存在22个单核苷酸多态性（SNP），其中13个位于内含子区，9个位于外显子区；位于最后一个外显子上SNP（G/A）的变异导致了编码氨基酸从缬氨酸变为异亮氨酸。MdPH1蛋白包含8个跨膜结构域，其中蛋白N端包含3个跨膜结构域，C端包含5个跨膜结构域。系统进化分析结果显示，苹果中的PH家族成员与梨（Pyrus communis L.）中的PH家族成员聚集成一簇。组织特异性表达结果发现，MdPH1基因在苹果果实中的表达量最高，其次是叶、花和根，茎中表达量最低。亚细胞定位分析表明MdPH1蛋白定位于液泡膜上。Abstract: In this study, the malic acid content in Malus fruit was determined using Malus ombrophila Hand.-Mazz, with comparative transcriptome analysis conducted among developmental stages. A candidate gene for fruit acidity, designated as MdPH1, was identified. Genomic sequencing and gene structure analysis showed that the cDNA sequence contained a 2829 bp open reading frame and encoded a 942 amino acid polypeptide. The genomic DNA of MdPH1 was 4269 bp in length and consisted of eight exons and seven introns. According to the PH1 gene sequence in 10 Malus accessions, 22 single nucleotide polymorphisms (SNPs) were identified in the genomic sequence. Of these SNPs, 13 were located in introns and nine were located in exons. Variation of a SNP (G/A) on the last exon resulted in the conversion of the encoded amino acid from valine to isoleucine. In addition, the MdPH1 protein contained eight transmembrane domains (TMDs). Of these TMDs, three were located in the amino terminal and five were located in the carboxyl terminal. Exploration of the phylogenetic relationships revealed a close relationship between PH1 genes from apple and pear. Gene expression indicated that MdPH1 was highly expressed in apple fruit, followed by the leaves and flower, and finally the stem. Subcellular localization assay showed that MdPH1 was localized to the tonoplast. Our study provides useful knowledge to better understand the complex mechanisms regulating apple fruit acidity.
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Keywords:
- Malus ombrophila /
- Fruit acidity /
- MdPH1 gene /
- Gene structure /
- Gene expression /
- Subcellular localization
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