砂梨2个内切-β-1,4-葡聚糖酶基因cDNA的克隆及其在果实贮藏过程中的表达分析  

Cloning Two Members of Endo-1,4-β-glucanases Gene Family from Sandy Pear and Their Expression Characteristics during Shelf Life

作　　者：丛郁[1] 李慧[1] 颜志梅[1] 俞明亮[1] 常有宏[1] 

机构地区：[1]江苏省农业科学院园艺研究所,江苏南京210014

出　　处：《江苏农业学报》2010年第2期383-389,共7页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省博士后基金项目(070213C);江苏省农业科学院博士后基金资助项目(6510613);国家农业科技成果转化基金项目(2008GB2C100100);江苏省科技基础设施建设计划项目(BM2008008)

摘　　要：为从分子水平上揭示EG基因在早熟砂梨软化过程中的作用机制,该研究以早熟砂梨品种翠冠果肉cDNA为模板,根据NCBI上登录的其他植物内切-β-1,4-葡聚糖酶(Endo-1,4-β-glucanas,EGases)基因编码区设计引物,利用RT-PCR技术,克隆内切-β-1,4-葡聚糖酶基因家族中的2个成员PpEG1和PpEG2的编码区序列,并在此基础上,利用半定量RT-PCR技术,探讨两基因在夏季货架期1-MCP处理条件下翠冠果肉软化过程中的表达情况。结果表明:PpEG1编码区包含1 869个核苷酸,编码1个由622位氨基酸组成的多肽,PpEG2编码区包含1 863个核苷酸,编码1个由620位氨基酸组成的多肽;PpEG1和PpEG2都含有糖基水解酶家族-9(Glycosyl-hydrolases-family-9)活性位点,但PpEG2比PpEG1的C端多1个碳水化合物结合组件(Carbohydrate-binding module,CBM);PpEG1与PpEG2分别位于分子进化树的2个不同发育分枝上;在室温货架期间,翠冠果肉中PpEG1的表达量先上升,贮藏4 d时其表达量最高,后缓慢下降,而PpEG2的表达量开始缓慢上升,在贮藏12 d时出现mRNA的积累高峰;1-MCP对PpEG1和PpEG2的表达不起延缓或促进作用。PpEG1与PpEG2在翠冠果实软化过程中的不同表达规律显示,PpEG1与PpEG2作用于不同的β-1,4-葡聚糖多聚分子底物,且PpEG1和PpEG2的表达均不受乙烯调控。The objective of this study was to illuminate the mechanism of EG（Endo-1,4-β-glucanas,EGases）genes in the sandy pear softening process through the gene cloning and expression regulation research of EG genes in Cuiguan pear.Two coding region sequences of EG gene were amplified form sandy pear（Pyrus pyrifolia Nakai） pulp cDNA by RT-PCR method,whose primers were designed on the basis of coding region of other EG genes submitted to GenBank.The expression regulation was researched by semi-quantitative RT-PCR during summer shelf life with or without 1-MCP treatment.PpEG1 cDNA has an open reading frame of 1 869 nucleotides and encodes a polypeptide including 622 residues,and relative molecular mass was 69 040;PpEG2 cDNA has an open reading frame of 1 863 nucleotides and encodes a polypeptide including 620 residues,and relative molecular mass was 68 250.Both PpEG1 and PpEG2 had glycosyl-hydrolases-family-9 active site;PpEG2 had a carbohydrate-binding module at the end of carboxyl terminal side.but PpEG1 did not.The phylogenetic tree analysis revealed that PpEG1 and PpEG2 were located at two different branchs.Duning high temperature storage（28-32 ℃）,the semi-quantitative RT-PCR results showed that expression abundance of PpEG1 reached the highest at day 4,then declined slowly,while the expression abundance of PpEG2 continued to rise slowly and reached the highest at day 12.Ethylene receptor inhibitor 1-MCP was used to prevent the sandy pear fruit softening,but neither PpEG1 nor PpEG2 was affected.The different expression regulations of PpEG1 and PpEG2 of Cuiguan pear indicated that PpEG1 and PpEG2 acted on different β-1,4-glucanases polymer substrates in the softening process and both of their expression were not affected by the ethylene regulation.

关 键 词：砂梨 内切--β1 4-葡聚糖酶 基因克隆 序列分析 表达特性 

分 类 号：Q785[生物学—分子生物学]