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[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。
[Objective] The aim of the study was to clone the full-length cDNA sequence of caffeoyl-CoA-methyltransferase (CCoAOMT) from the fruit of Pyrus bretschneideri ’Dangshansuli’ and analyze its bioinformatics and gene expression differences , Explained its role in lignin biosynthesis pathway in pear fruit, with a view to providing some theoretical basis for the future use of gene regulation technology to change the content of stone cells in fruit so as to improve the quality of pear fruit. [Method] With 15-year-old ’Dangshan Su pear’ fruit as test material, the sequence alignment of plant lignification CCoAOMT gene sequence and pear genome database was searched by searching from NCBI database and cloned by RT-PCR A key enzyme in the lignin biosynthesis pathway, PbCCoAOMT, was deposited with GenBank accession number KJ577544. [Result] The full length of this gene was 1133 bp with a complete 744 bp open reading frame (ORF) encoding 247 amino acids. Sequence alignment and phylogenetic tree analysis showed that the CCoAOMT enzyme is an Oxymethyltransferase that has high similarity with other plant CCoAOMT protein sequences, especially the highest similarity with loquat (98.79%) recent. Fluorescence quantitative PCR analysis of its expression patterns in different developmental stages of fruit showed that gene expression increased first and then decreased, similar to that of pear fruit. [Conclusion] It was preliminarily believed that the PbCCoAOMT gene cloned from ’Dangshan Pear’ might be involved in the metabolism of lignin in pear fruit.