新疆野苹果种子MsPYR1-like基因克隆、表达与亚细胞定位分析

The PYR/PYL/RCAR gene family,as abscisic acid(ABA)receptors,is one of the three major components involved in early ABA sensing and signaling,and the regulation of seed dormancy is mainly achieved by regulating the metabolic and signaling pathways of gibberellin(GA)and abscisic acid(ABA).In this study,the MsPYR1-like gene of Malus sieversii seeds was cloned,subcellularly localized and expressed to provide a theoretical basis for its involvement in the mechanism of seed dormancy release in Malus sieversii seeds.Based on the transcriptome database of Malus sieversii seeds,the PYR1-like family gene sequences were obtained,the PYR1-like gene(tentatively named MsPYR1-like)was cloned and bioinformatically analyzed,and the encoded protein was subjected to sequence characterization and evolutionary tree,vector construction for subcellular localization.Using the low temperature lamination method,seeds were evenly mixed with wet sandy soil at a volume of 1:3 and placed in plastic boxes,stored in a refrigerator at 4°C,and seed humidity was maintained.Seeds were selected for 0,30,60,90,and 120 d.The gene expression pattern of MsPYR1-like in Malus sieversii seeds at different stratification periods by quantitative real-time fluorescence(qRT-PCR)analysis.Showed that the full length of the open reading frame of the obtained MsPYR1-like gene was 621bp,encoding 206 amino acids,and the encoded protein sequence had a structural domain of the PYR/PYL/RACR protein family with a hydrophobic ligand-binding deep pocket-like structural domain that specifically binds ABA and mediates its signaling.The physicochemical analysis of the encoded protein showed that the molecular weight of the encoded protein was 23.14 kDa;the predicted isoelectric point pI was 5.37;the instability coefficient was 41.16,which was classified as an unstable protein;the aliphatic amino acid index was 74.17,which was thermally stable;and the grand average hydrophobicity index was-0.557,which was a hydrophilic protein.With 33 negatively charged residues aspartic acid and glutamic acid(Asp + Glu)and 24 positively charged residues arginine and lysine(Arg + Lys);in the composition of amino acid residues,valine(Val)was high,up to 9.7%,while hydrophilic amino acids such as glutamic acid(Glu),leucine(Leu),aspartic acid(Asp)and serine(Ser)The content of hydrophilic amino acids such as Glu,Leu,Asp and Ser was also relatively high.The transmembrane structure prediction analysis did not classify it as a transmembrane protein.It was hypothesized that MsPYR1-like protein may be localized in the cytoplasm and is a receptor for the cytoplasm by the online prediction software Plant-mPLoc.The subcellular localization results showed that the protein was localized in the nucleus and cytoplasm of the cell.Secondary structure prediction of the MsPYR1-like protein showed that it consists of 35.44%ɑ-helix,3.88%β-turn,15.05%extended chain and 45.63%irregular coiling.The primary structure of MsPYR1-like protein was derived as α-helix and irregular coiling,and the overall secondary structure of the protein consisted of a structure favoring protein stability.The tertiary structure predicted that the MsPYR1-like protein had a high similarity to the template at amino acids 13-204 with 76.04%similarity.Evolutionary analysis showed that the obtained MsPYR1-like is closely related to the European wild apple homologue,with a homology rate of 98%.The MsPYR1-like protein is identical in length to the European wild apple protein,encoding 206 amino acids,but differs in three amino acids,with a high sequence similarity of 98%and the next highest similarity with lentil(94%).The MsPYR1-like protein has high sequence identity with different plant PYR1 proteins,indicating that the gene is more conserved in the evolutionary process.qRT-PCR results showed that the expression of the MsPYR1-like gene was significantly down-regulated at 30d of low temperature lamination,consistently decreased at 30-90d of lamination,reached a minimum at 90d of lamination and The expression of MsPYR1-like gene was significantly down-regulated at 30d of low-temperature lamination,decreased from 30 to 90d of lamination,reached a minimum at 90d of lamination,and was up-regulated from 90 to 120d of lamination.The transcriptome data showed that the expression of MsPYR1-like gene was highest at 30 d of lamination,and the expression showed down-regulated expression during the lamination process.The expression of transcriptome and fluorescence quantification both showed down-regulated expression with increasing lamellar time,which showed the same trend of change.MsPYR1-like in Malus sieversii seeds has an important regulatory role in regulating the dormancy release of seeds.The above study illustrates the dormancy mechanism of Malus sieversii seeds,and provides a theoretical basis for understanding the subsequent functional analysis studies in Malus sieversii seeds.