【作者】 卞伟华；

【导师】 江昌俊；

【作者基本信息】 安徽农业大学 ， 遗传学， 2005， 硕士

【摘要】 植物中含有丰富的 β-半乳糖苷酶,它在植物各个时期的含量对于植物的成熟、风味、颜色及采收后加工有重要意义。本论文首次从桃树中克隆出 β-半乳糖苷酶基因cDNA 全长序列,并尝试在大肠杆菌中进行初步表达。这将有助于弄清果蔬成熟和风味形成的机制,为进一步运用生物技术对其进行有利的调控及生产广受消费者欢迎的果蔬产品打下基础。生产的植物来源的 β-半乳糖苷酶可以更安全、更可靠地运用于食品工业、医药工业、免疫和环境保护等领域。 根据桃树叶片生化特点,应用 Chomcznski 一步法,并作了部分修改,从鲜叶中分离、提取总 RNA。利用 GenBank 中已经登录的其它植物中该酶基因的保守序列,设计简并引物,采用 RT-PCR 技术,扩增出 387bp 的 cDNA 片段。将扩增片段克隆到pMD18-T 载体上进行序列测定和分析,结果表明,此片段的核苷酸序列与其它植物 β-半乳糖苷酶基因 cDNA 相应序列有很好的同源性,推断该 PCR 特异产物可能为桃树β-半乳糖苷酶基因 cDNA 片段。 以此特异片段设计特异引物,采用 RACE 技术分别获得了 β-半乳糖苷酶基因cDNA3’端 2343bp 和 5’端 840bp 片段,再依据上述两端序列设计一对引物,扩增出cDNA 全长序列为 3285bp。经 Blast 搜索表明:克隆所得碱基序列与已克隆出的沙梨、番茄、葡萄、拟南芥、鹰嘴豆、绿豆等植物体内 β-半乳糖苷酶基因的 cDNA 序列有80%以上的同源性,我们推断所得到的序列可能为桃树中 β-半乳糖苷酶基因 cDNA 的全长序列。利用分子生物学软件对该序列进行分析,结果表明,该 cDNA 包含 1 个2559bp 的开放阅读框,编码 853 个氨基酸。3’端非编码区 539 bp,翻译起始于第 185个碱基,终止于第 2746 个碱基。 根据上述 β-半乳糖苷酶基因 cDNA 全长序列设计引物,PCR 扩增该 cDNA 编码区序列。将其插入到大肠杆菌表达载体 pET-32a(+)中,构建原核表达载体。转化BL21trxB(DE3),筛选重组菌株。经 IPTG 诱导表达后,SDS-PAGE 呈现约 115kDa 特异表达带,但与专一性底物邻硝基苯酚-β-D-半乳糖苷(O-nitrophenyl-β-D-galactoside,ONPG)反应,表达产物没有 β-半乳糖苷酶活性。更多还原

【Abstract】 There are abundant beta-galactosidases in plant. The different content in everyperiod take a very important part in mature, flavor, colour and manufacture after plucked.In this paper, the complete cDNA sequence of beta-galactosidase was cloned in the peachleaf at the first time and it was expressed in E.coli. This contributed to make clear themechanism of the mature of fruits and vegetables as well as of the formation of flavor. Italso established the foundation that using biotechnology to produce the popular fruits andvegetables. The beta-galactosidase from plant can be applied to food, medicine, immunity,environment protection etc. Total RNA from young leaves of peach was extracted by chomczynsk’ single stepmethod of RNA isolation, which was partially modified on the basis of the features ofbiochemistry composition in fresh peach leaves. The degenerate primers were designedand synthesized according to the highly conservative sequences among the knownbeta-galactosidase genes. A cDNA fragment of 387bp was amplified by RT-PCR, whichwas subsequently cloned into pMD18-T Vector for sequencing analysis. The result ofsequencing analysis showed that the nucleotide and deduced amino acids sequences hadhigh identity with the published corresponding parts of beta-galactosidase cDNAs of otherplants. The results indicated that the special PCR product was probably a cDNA fragmentof beta-galactosidase in peach. Two special primers of RACE were designed based on the above sequence of theRT-PCR product. Two fragments of 5’cDNA (840bp) and 3’cDNA (2343bp) wereamplified by RACE reaction. Two complete sequence primers were designed based on theresult of RACE. A 3285bp sequence was amplified by PCR. By the Blast programcomparison, the analysis of the product showed that the nucleotide shared more than 80%homologous to the corresponding parts of beta-galactosidase gene family of Pyraspyrifolia, Lycopersicon esculeutum, Arabidopsis thaliana, Cicer arietinum, Vigna radiateetc. The results indicate that 3285bp sequence is probably complete cDNA fragment ofbeta-galactosidase in peach. The nucleotide sequence was analyzed by the molecularbiology software, The results shows that the open reading frame is 2559bp which encodes853 amino acids(AA). The 3’UTR is 539bp. The translation is from the 185th base to the2746th base.The primers were designed according to the complete cDNA having been sequenced.The open reading frame cDNA was amplified by the methods of PCR. Then it wasconstructed into the vector pET-32a(+) designed for recombinant strain. After IPTGinduction, it was observed that there was a special band about 115kDa on SDS-PAGE. Butthere was no beta-galactosidase activity when assayed using ONPG as substrate.更多还原