‘砀山酥梨’果实CCoAOMT基因的克隆与表达分析

Cloning and expression analysis of CCoAOMT gene in fruit of ‘Dangshansuli'

[目的]通过克隆‘砀山酥梨’(Pyrus bretschneideri ‘Dangshansuli’)果实中咖啡酰-辅酶AO-甲基转移酶(CCoAOMT)基因的全长序列,并对其进行生物信息学及基因表达差异分析,阐释了其在梨果实木质素合成途径的作用,以期为今后利用基因调控技术来改变果实中石细胞含量从而改善梨果实品质提供一定的理论依据。[方法]以15年生‘砀山酥梨’果实为试材,根据从NCBI数据库中搜索得到关于植物木质化的CCoAOMT基因序列与梨基因组数据库调取的序列比对,利用RT-PCR技术克隆得到了木质素生物合成途径中的一种关键酶基因PbCCoAOMT,GenBank登录号为KJ577544。[结果]该基因全长1133bp,具有1个744bp的完整开放阅读框(ORF),编码247个氨基酸。序列比对及系统进化树分析表明:CCoAOMT酶是氧甲基转移酶类,与其他植物CCoAOMT编码的蛋白序列有较高的相似性,尤其与枇杷的相似度最高,达到98.79%,且亲缘关系最近。利用荧光定量PCR技术对其在果实不同发育时期表达模式的分析发现,基因表达量呈先上升后下降的趋势,与梨果实中石细胞含量的变化趋势相似。[结论]初步认为从‘砀山酥梨’中克隆得到的PbCCoAOMT基因可能参与了梨果实中木质素的代谢。

[Objectives]Lignin is widely distributed in plant cell walls and confers strength,rigidity and impermeability,which are the principal components of stone cells in pear. Caffeoyl-CoA O-methyltransferase（ CCoAOMT） plays an important role in the biosynthesis of lignin,and the 3-O-methylation of caffeoyl CoA leading to the formation of feruloyl Co A is catalyzed by the enzyme CCoAOMT.[Methods]In order to investigate the function of CCoAOMT in the synthesis of pear stone cells,15-year-old trees of Pyrus bretschneideri‘Dangshansuli＇were used in this study,and we cloned Pb CCoAOMT gene and analyzed its biological information,and studied the gene expression at different stages during pear fruit development. These results contributed to providing some theoretical reference and practical basis to improve the quality of pear. Comparing CCoAOMT gene sequences associated with lignification obtained from from the NCBI database with pear genome database,the Pb CCoAOMT gene cDNA sequence involved in lignin biosynthesis was cloned by RT-PCR techniques. Gene structure analysis relied on GSDS. Sequence alignments and homology sequence searchers in databases were carried out using NCBI BLAST webpage. Deduced amino acid sequences were obtained by Primer Premier 5.0 software. Phylogenetic relationships among sequences were performed with MEGA 6.06. The molecular structure and physicochemical properties of proteins were analyzed by Prot Param. Hydrophobic analysis of deduced protein sequences was demonstrated by means of Prot Scale. Signal peptide of deduced protein sequences was predicted by Bio XM 2. 6. The transmembrane domain of deduced protein sequences was predicted by TMPRED. Secondary and tertiary structures of deduced protein sequences were aligned by SOPMA and SWISS-MODEL separately. For gene expression analysis,total genomic RNA was isolated from fruit samples collected at different stages during pearfruit development according to the CTAB method and c DNAs were generated following the manufacturers＇ recommendations to a final volume of 20 μL,gene quantifications were performed according to the manufacturers＇ instructions and Pyrus EFα1（ EFα1,accession No.： AY338250） was used as internal gene to evaluate the RT-qPCR assays. [Results]The full-length cDNA of Pb CCoAOMT（ Gen Bank accession No.： KJ577544） in pear was cloned. The sequence consists of 1 133 bp with an open reading frame of 744 bp,which encodes a polypeptide of 247 amino acids residues. Sequence alignment and phylogenetic analysis showed that CCoAOMT is a member of O-methyltransferases family and shared high similarity with CCoAOMT proteins from other species,especially with the highest similarity of 98.79% and phylogenetic relationship compared with the loquat. Real-time PCR results showed that the change of Pb CCoAOMT expression level was similar to that of stone cell content during fruit development,which showed a rise-down tendency.[Conclusions]Pb CCoAOMT gene was firstly isolated and characterized from pear,which may be involved in metabolism of lignin.