Cloning of SoDIPPR gene of pentatricopeptide repeat (PPR) protein family in potato and analysis of expression characteristics under drought conditions

Chino. PPR（pentatricopeptide repeat）蛋白家族在植物的生长发育、细胞器形成、细胞质雄性不育的育性恢复、RNA的编辑加工、细胞核与细胞器之间信号传递、逆境防御等方面具有重要作用。研究旨在了解PPR蛋白家族SoDIPPR基因在马铃薯中的表达情况及其在干旱胁迫下的作用。以抗旱马铃薯二倍体品系H145为材料，利用cDNA-AFLP技术寻找与抗旱性密切相关的基因cDNA片段，利用RACE技术克隆其基因全长cDNA，并利用半定量RT-PCR和Northern杂交法研究其在干旱条件下的表达情况。从抗旱马铃薯二倍体品系H145中克隆了一个836 bp全长cDNA序列，命名为SoDIPPR，该序列开放阅读框长为588 bp，编码195个氨基酸序列。序列分析表明，SoDIPPR蛋白存在PPR结构域、激酶结合区、和C端SMR（small MutS related protein）区域。SoDIPPR蛋白序列与GenBank数据库中的其它PPR蛋白进行同源序列比对，构建系统进化树，发现SoDIPPR 与其它14个高等植物的PPR蛋白同源性为57%～82%，与苜蓿DNA错配修复蛋白MuTs2、水稻盐诱导蛋白（Q9LS25）和叶绿体RNA结合蛋白（Q75IP8）的同源性达69%～82%。半定量RT-PCR和Northern杂交结果表明，SoDIPPR基因在干旱胁迫下叶片和根系里的表达量明显增加，说明SoDIPPR基因在马铃薯抗旱中起一定作用。在持续干旱条件下，SoDIPPR基因在抗旱品系H145与干旱敏感品系H214中表达模式不同。马铃薯SoDIPPR基因编码的蛋白与其它植物PPR家族蛋白同源性较高，SoDIPPR基因参与马铃薯对干旱胁迫的应答反应，可能对抵抗干旱胁迫有一定作用。

Inglés. Some pentatricopeptide repeat (PPR) proteins are involved in plant development, organelle biogenesis, restoring of cytoplasmic male sterilities, RNA processing and editing in mitochondria and chloroplasts, plastid-to-nucleus retrograde signaling, and responses to environmental stresses. The objectives of the present study were to clone the SoDIPPR gene of PPR protein family in potato and analyze its expression under drought conditions. The potato (Solanum tuberosum) genotype H145 that is diploid of drought tolerance was used as a material to isolate differentially expressed genes related to drought Ctolerance by cDNA-AFLP and clone the full-length cDNA of SoDIPPR by rapid amplification of cDNA ends (RACE). Its temporal expression pattern was studied by semi-quantitative RT-PCR and northern blot analysis. On the basis of one cDNA-AFLP differential fragment isolated from induced mRNA in the potato genotype H145, the 836 bp full length cDNA of a putative PPR family gene, SoDIPPR gene was cloned by RACE. The gene consisted of 588 bp encoding a hypothetical protein of 195 amino acids. Sequence analysis revealed that it has PPR motifs, a kinase site, a small mutS related (SMR) domain found in the SoDIPPR protein. Alignment and phylogenetic analysis of the amino acid sequence deduced from the fragment and the PPR protein family sequences of other plants retrieved from GenBank were carried out by ClustalX program, which showed that the sequences homology of reported 14 PPR proteins from other plants with SoDIPPR was among 57%-82%. SoDIPPR shares high identity (69%-82%) with mismatch repair protein MutS2 of alfalfa, salt-inducible protein of rice and chloroplastic RNA-binding protein of rice. The level of transcript of SoDIPPR was investigated by semi-quantitative RT-PCR and northern blot. The result showed that the expression of SoDIPPR was significantly enhanced under drought. The different profiles of expression of SoDIPPR gene were detected under continuous drought between potato drought tolerance genotype H145 and H214 which is susceptive to drought. Deduced protein of potato SoDIPPR gene has higher homology with PPR proteins of other plants. The results indicate that SoDIPPR gene may play an important role in response to drought stress in potato.