【作者】 刘海峰；

【导师】 王军； 刘关君；

【作者基本信息】 东北林业大学 ， 森林植物资源学， 2010， 博士

【摘要】 山葡萄(Vitis amurensis Rupr.)是我国重要的野生果树资源。花色苷是山葡萄果实中主要的多酚类次生代谢产物,与山葡萄的抗病性、色泽、风味等密切相关。花色苷不仅影响植物源食品的感官和营养价值,还具有多种医药方面的生理活性。本文进行了山葡萄转色期果皮cDNA文库的构建,对山葡萄花色苷生物合成相关结构基因进行了克隆和序列分析,对山葡萄果实不同成熟期,不同组织与器官中花色苷合成的相关基因表达进行了初步研究。主要研究结果如下：通过三种提取方法提取山葡萄果皮和叶片中的总RNA,其中改良CTAB法能分离出高质量的RNA,可用于下一步的分子生物学研究。以转色期(50%着色)山葡萄果皮为材料,提取总RNA合成cDNA,连接到质粒载体pDNR-LIB上,采用电穿孔法将重组质粒转化到DH5a中,成功构建了山葡萄果皮cDNA文库。文库质量鉴定表明,原始文库滴度为1.12×106 pfu·mL-1,扩增后的文库滴度为2.82×109 pfu·mL-1,重组率接近100%,插入片段大小范围在0.5-2 kb之间,平均约为0.80 kb,表明应用SMARTTM技术已成功构建了山葡萄转色期果皮cDNA文库。山葡萄转色期果皮cDNA文库测序获得974条有效EST序列。GenBank登录号：GW666520-GW667493。974条EST拼接后代表了710个UnigeneS。BLAST注释结果表明,具有已知或推测功能基因270个,未知功能基因258个,未比对上的基因182个(可能是新基因)。根据BLASTX注释与GO分类,199条EST序列具有细胞组件功能,313条EST序列在分子功能上得到注释,341条EST序列在生物学途径上得到注释。采用RT-PCR、SMARTTM RACE技术从山葡萄成熟期果皮中克隆到了类黄酮-3’-羟化酶(VAmF3’H)、类黄酮-3’,5’-羟化酶(VAmF3’5’H)、黄烷酮醇4-还原酶(VAmDFR)、无色花色素双加氧酶(VAmLDOX)、类黄酮3-O-葡萄糖基转移酶(VAm3GT)、O-甲基转移酶(VAmOMT)、谷胱甘肽转移酶(VAmGST4)和类黄酮5-O-葡萄糖基转移酶(VAmOGT)等8个基因的cDNA全长序列。GenBank登录号为：FJ645766、FJ645767、FJ645768、FJ645769、FJ169463、GU237132、FJ645770和GU237133。利用生物信息学分析,推测8个基因分别编码509、508、337、355、456、235、213和448个氨基酸的多肽,分子量为56.14、56.70、37.50、40.19、50.19、26.35、24.24和49.84 kDa。与其它物种相关基因的氨基酸序列进行了多重比较,各基因与欧亚种葡萄(Vitis vinifera L.)相关基因的相似性均超过90%。在此基础上构建了这8个基因与其它物种相应基因的分子进化树,分析了与其它物种相关基因的亲缘关系。各基因信号肽预测结果表明,8个基因都不具有信号肽结构,对各基因分别进行了疏水性分析,初步预测了其二级结构。对山葡萄幼叶、成熟叶、茎、果肉和果皮中花色苷生物合成结构基因的表达进行了初步研究。半定量RT-PCR分析结果表明,VAmF3’H和VAmF3’5’H在花后各个时期的果皮中均有表达,转色期上调表达；VAmDFR、VAmLDOX和VAmOGT在花后各个时期的果皮中均有表达,各个时期的表达强度差异不明显；VAm3GT和VAmGST4在果实转色期前基本不表达,转色期为上调表达；VAmOMT在花后两周有少量表达,随后表达量下降,转色期开始上调表达。VAmF3’H、VAmF3’5’H、VAmDFR、VAmLDOX和VAmOGT在各个组织与器官中均有表达,但VAmF3’5’H在成熟叶中的表达量很少；VAm3GT、VAmOMT和VAmGST4在叶片中都不表达,在茎、果肉、果皮中都有表达,VAmOMT在果肉中表达丰度低。Northern杂交结果表明,VAm3GT在果实转色期前基本不表达,转色期上调表达;VAmOGT在果实成熟过程中均有表达。用DNA重组技术构建了PQE30-Xa-VAm3GT和PQE30-Xa-VAmOGT原核表达载体,转入大肠杆菌M15表达6×His融合蛋白。结果表明,重组蛋白进行了大量表达,通过Ni-Sepharose FF纯化后,两种蛋白的分子量为50 kDa和49.5 kDa左右,重组蛋白的酶学功能和特性正在进行研究。更多还原

【Abstract】 Vitis amurensis is an important wild fruit resource. Anthocyanins are the major polyphenol secondary metabolites in V. amurensis, and which are closely related to disease resistance, color, flavor and other indicators. Anthocyanin not only affects the sensory and nutritional value of plant foods, but also has a variety of physiological activity of medicine. In this paper, we constructed V amurensis veraison skin cDNA library, cloned and sequenced the structural genes of V. amurensis anthocyanin biosynthesis. Anthocyanin biosynthesis related gene expression in different V. amurensis tissues, organs and various period of berry were preliminary studied. The major findings are as follows:RNA was isolated from V. amurensis skins and leaves using three methods of extraction. Highly-grade RNA was isolated by using modified CTAB method and suitable to the demands of further molecular biological research. The veraison skins (50% colored) of V. amurensis was used as experimental material, from which the total RNA was extracted. The cDNA was synthesized and the ds cDNA fragment was ligated into the pDNR-LIB vector. The recombinant plasmid was transformed into E.coli DH5a by electroporation. The library qualification evaluation showed:the titer of primary cDNA library was 1.12×106 pfu·mL-1, the titer of amplified library was 2.82×109 pfu·mL-1, the percentage of recombination was about 100%, the fragment size of inserted was 0.5-2 kb. The average insert size was 0.80 kb. The result showed that the cDNA library of V. amurensis veraison skin was constructed successfully with SMARTTM technology.A total of 974 high quality ESTs were obtained from cDNA library of V. amurensis veraison skins. The GenBank accession numbers are GW666520-GW667493. The 974 ESTs were assembled into 710 unigenes. The results of BLAST annotation showed:270 known or putative functional gene,258 unknown gene,182 no hits (maybe new genes). Based on the BlastX annotation and GO analysis,199 ESTs have the cellular component function,313 ESTs were associated with molecular functions, and 341 ESTs with biological process.The full-length cDNA sequences of VAmF3’H, VAmF3’5’H, VAmDFR, VAmLDOX, VAm3GT, VAmOMT, VAmGST4 and VAmOGT genes in V. amurensis were cloned by the technology of RT-PCR and SMARTTM RACE. The GenBank accession number is FJ645766, FJ645767, FJ645768, FJ645769, FJ169463, GU237132, FJ645770, and GU237133. Using bioinformatics analysis, speculated that 8 genes encode 509,508,337,355,456,235,213 and 448 amino acids, with molecular mass of 56.14,56.70,37.50,40.19,50.19,26.35,24.24, and 49.84 kDa. With multiple comparisons to the amino acid sequence of others species related genes, the similarity of each gene and related genes of V. vinifera is more than 90%. On this basis, molecular evolutionary tree of these eight genes and the corresponding genes of other species was constructed, the genetic relationship with related genes of other species was analyzed. Each gene signal peptide prediction results show that the eight genes do not have a signal peptide structure, the various genes were carried out hydrophobicity analysis, preliminary forecast of their secondary structure.Expression of anthocyanin biosynthetic structure genes inⅤ. amurensis young leaves, mature leaves, stems, pulp and skin were preliminary studied. The results of semi-quantitative RT-PCR showed that the expression of VAmF3’H and VAmF3’5’H happened in various period of the skins after flowering and up-regulated expression in veraison. VAmDFR, VAmLDOX and VAmOGT are expressed in each period of skins after flowering, whereas the expression intensity of each period was not significantly different. VAm3GT and VAmGST4 almost have no expression before veraison, and up-regulated expression in veraison period. The expression quantity of VAmOMT is small in two weeks after flowering, then the expression declined and increased at veraison. VAmF3’H, VAmF3’5’H, VAmDFR, VAmLDOX and VAmOGT were expressed in various tissues and organs. However, VAmF3’5’H is expressed in mature leaves at a low level. VAm3GT, VAmOMT and VAmGST4 are expressed in stems, pulp and skins, not in leaves. VAmOMT is expressed in low abundance in the pulp. Northern blot results showed that VAm3GT almost have no expression before veraison, up-regulated expressed at veraison. VAmOGT is expressed at berry maturating.PQE30-Xa-VAm3GT and PQE30-Xa-VAmOGT prokaryotic expression vector was constructed by DNA recombination technology, which was transformed into E.coli 15 expressing 6×His fusion protein. The results showed that the recombination protein expresses at a high level. The molecular mass of two protein purified by Ni-Sepharose FF is 50 kDa and 49.5 kDa. Recombinant protein enzymatic functions and features are being studied.更多还原