| 摘要: |
| 【目的】克隆新疆野苹果（Malus sieversii）脱落酸受体基因MsPYR1-like，研究其在4 ℃低温沙藏过程中的表达规律，为揭示MsPYR1-like基因的功能及其在新疆野苹果种子休眠解除中的作用提供依据。【方法】从NCBI数据库中选择金冠苹果（Malus domestica）PYR1-like基因序列，将其与新疆野苹果转录组数据库进行BLAST同源比对，获得新疆野苹果PYR1-like基因序列。采用在线网站Expasy ProtParma、Expasy ProtScale、TMHMM2.0、SignalP-6.0和Plant-mPLoc，对MsPYR1-like蛋白进行理化特性和亲疏水性分析以及跨膜结构、信号肽和亚细胞定位预测。使用NCBI的CDD数据库、SOPMA和Swiss-Model Workspace，分析MsPYR1-like蛋白的保守结构域、预测蛋白二级和三级结构。利用DNAMAN和MEGA 11进行蛋白序列比对和构建蛋白系统进化树，构建MsPYR1-like基因载体并对侵染烟草叶片进行亚细胞定位。采用室温和4 ℃低温沙藏层积法处理新疆野苹果种子，于处理0，30，60，90和120 d时取样测定种子萌发率，同时采用实时荧光定量PCR检测种子MsPYR1-like的相对表达量，采用酶联免疫吸附法测定脱落酸含量，分析种子萌发率、MsPYR1-like相对表达量和脱落酸含量三者之间的关系。【结果】MsPYR1-like基因开放阅读框全长621 bp，编码206个氨基酸，分子质量为23.14 ku，等电点5.37，不稳定系数41.16，脂肪族氨基酸指数74.17，为亲水性蛋白，不含信号肽和跨膜结构。其蛋白序列具有SRPBCC 超家族结构域，属于PYR/PYL/RACR蛋白家族，α-螺旋和无规则卷曲为二级结构主要组成。MsPYR1-like亚细胞定位在细胞核和细胞质膜中。同源性分析和系统进化树结果显示，MsPYR1-like与欧洲野苹果（Malus sylvestris）同源蛋白的亲缘关系最近。室温处理不同时间的新疆野菜果种子萌发率均为0%，低温沙藏0，30，60，90，120 d的种子萌发率分别为0%，0%，7%，95%和100%；随沙藏层积时间的增加，室温和4 ℃低温处理新疆野苹果种子的脱落酸含量均呈下降趋势，室温处理MsPYR1-like基因的相对表达量逐渐增加，而低温沙藏处理MsPYR1-like基因的相对表达量呈下降趋势。【结论】MsPYR1-like基因可能参与了新疆野苹果种子的休眠解除。 |
| 关键词: 种子休眠 种子萌发 脱落酸 新疆野苹果 MsPYR1-like 生物信息学 |
| DOI：10.13207/j.cnki.jnwafu.2025.04.003 |
| 分类号: |
| 基金项目:新疆维吾尔自治区自然科学基金面上项目（2023D01A28）；新疆农业大学高层次人才引进项目（2521GCCRC） |
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| Bioinformatic analysis of MsPYR1-like gene in Malus sieversii and its expression pattern during seed germination |
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FANG Zhen, LI Jing, MA Juan, ZHANG Kai, YE Chunxiu
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College of Forestry and Landscape Architecture,Xinjiang Agricultural University,Urumqi,Xinjiang 830052,China
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| Abstract: |
| 【Objective】The MsPYR1-like gene of abscisic acid receptor in Malus sieversii was cloned and its expression pattern under low-temperature sand storage stratification of 4 ℃ was analyzed to provide a basis for revealing the function of MsPYR1-like gene and its involvement in the role of seed dormancy lifting.【Method】The golden delicious apple (Malus domestica) PYR1-like gene sequence was selected from the NCBI database.The PYR1-like gene sequence of Malus sieversii was obtained by BLAST homology comparison with the M.sieversii transcriptome database.Physicochemical properties,hydrophilicity analysis,transmembrane structure prediction,signal peptide and subcellular localization prediction of MsPYR1-like proteins were performed with the online websites Expasy ProtParma,Expasy ProtScale,TMHMM2.0,SignalP-6.0 and Plant-mPLoc.NCBI’s CDD database,SOPMA and Swiss Model Workspace were used to analyze the conserved structural domains of MsPYR1-like protein and to predict protein secondary and tertiary structures.DNAMAN and MEGA 11 were used to perform protein sequence comparison and construct protein phylogenetic tree.MsPYR1-like gene vector was constructed,and subcellular localization was performed on infiltrated tobacco leaves.The seeds of M.sieversii were treated by storage at room temperature and low temperature sand storage stratification of 4 ℃,and samples were taken at 0,30,60,90 and 120 d to determine the seed germination rate,and the relative expression of MsPYR1-like was detected by real time fluorescence PCR at the same time.The abscisic acid content was measured by enzyme linked immunosorbent assay,so as to analyze the relationship between seed germination rate,relative expression of MsPYR1-like and abscisic acid content.【Result】The results showed that the open reading frame of MsPYR1-like gene was 621 bp in length,encoding 206 amino acids,with a molecular mass of 23.14 ku,isoelectric point of 5.37,instability coefficient of 41.16,and aliphatic amino acid index of 74.17.It was a hydrophilic protein,with no signal peptide or transmembrane structure.Its protein sequence had the structural domains of SRPBCC superfamily,which belonged to the PYR/PYL/RACR family,with α-helix and random coil as the main components of the secondary structure.MsPYR1-like subcell was localized in the nucleus and plasma membrane.The results of homology analysis and phylogenetic tree showed that MsPYR1-like was most closely relative to the M.sylvestris homologous protein.The abscisic acid content tended to decrease with increasing stratification time.The seed germination rate was 0% at different time points of room temperature treatment,and 0%,0%,7%,95% and 100% at 0,30,60,90 and 120 d of low temperature sand storage,respectively.The abscisic acid content of M.sieversii seeds showed a decreasing trend with the increase of the sand storage layer time in both room temperature and low-temperature treatments of 4 ℃,and the relative expression of MsPYR1-like gene increased gradually in room temperature treatment,while the relative expression of MsPYR1-like gene showed a decreasing trend in low-temperature sand treatment.【Conclusion】The MsPYR1-like gene may be involved in the dormancy release in M.sieversii seeds. |
| Key words: seed dormancy seed germination abscisic acid Malus sieversii (Ledeb.) M.Roem. MsPYR1-like bioinformatics |
