【作者】 刘军；

【导师】 马锋旺；

【作者基本信息】 西北农林科技大学 ， 果树学， 2010， 硕士

【摘要】 抗坏血酸(L-ascorbic acid,AsA),又叫维生素C,对人类的健康起着重要作用,在植物的生理生化反应中也担当着至关重要的作用,尤其在植物处于逆境胁迫时。因此,对抗坏血酸的研究有着重要的意义。D-半乳糖醛酸还原酶基因,即GalUR基因,是可能的抗坏血酸合成途径——糖醛酸途径中的一个关键酶,通过对该基因的研究,能够为这种可能的交替途径提供分子生物学上的理论依据。本试验以美味猕猴桃品种‘秦美’为材料,克隆GalUR基因,并构建了各种表达载体,为进一步研究该基因的功能奠定了基础。本试验的主要研究结果如下:1.克隆得到了美味猕猴桃‘秦美’GalUR基因cDNA全长。该基因cDNA全长1028bp,含有990bp的完整开放阅读框,编码了329个氨基酸。该基因与Genebank中已登录的美味猕猴桃(FG475088.1)的同源性达到100%,与美味猕猴桃‘秦美’GalUR2(GU339038.1)的同源性为82%,与北柴胡的同源性为76%,与无梗花栎的同源性为79%。2.构建了猕猴桃GalUR基因的过量植物表达载体pWR/GalUR,并成功地转化根癌农杆菌EHA105感受态细胞。3.成功构建了猕猴桃GalUR基因干扰表达载体pHGRV/GalUR。同时采用改良冻融法,将其转入根癌农杆菌EHA105感受态细胞。4.从猕猴桃果实的不同发育时期以及猕猴桃不同组织两个方面检测与AsA含量有关的一系列相关指标及GalUR基因的表达水平的变化。结果发现,猕猴桃果实花后30d,AsA含量达到最大值,此时GalUR基因的表达量也达到最高值;然后AsA含量趋于稳定,GalUR基因mRNA水平的相对表达量变化也不大,这表明GalUR基因与AsA的合成有着密不可分的关系。在猕猴桃成熟叶中的与AsA含量相关的各个指标明显高于其他组织,GalUR基因在成熟叶中的转录水平也高于其他组织;但叶柄中的AsA含量最低,其中的GalUR基因表达水平却与成熟叶差异不明显,果柄与此相反。5.将GalUR基因的编码序列按照正确阅读框插入到原核表达载体pET-32a(+)中,构建了原核重组表达载体pET/GalUR。将重组表达载体转入宿主菌E.coli BL21后,经IPTG诱导后成功地表达出一条约57kD大小的特异蛋白带,可溶性分析表明该蛋白主要以包涵体形式存在;并优化其表达条件,经SDS-PAGE电泳显示,在0.01mM的IPTG诱导4、5h时蛋白表达量最大,在22、30℃条件下,重组蛋白中的可溶性蛋白含量较高。用切胶法免疫家兔制备了抗GalUR融合蛋白的多克隆抗体。6.分别提取猕猴桃幼叶、成熟叶、叶柄、幼果、成熟果、韧皮部的总蛋白,用制备的多克隆抗体进行western blot分析。在约37kD大小的位置处有一条特异性蛋白条带,符合预期结果;在成熟叶片中,GalUR蛋白的表达量最高,其次是幼叶、叶柄、幼果、韧皮部,成熟果中最低,与定量分析结果基本相符。根据western blot分析说明制备的多克隆抗体能够特异识别猕猴桃植物体内的抗原。更多还原

【Abstract】 L-ascorbic acid (AsA) which is generally called Vitamin C by people plays essential roles in maintaining human health and improving plant biochemistry reaction, especially when plants are in adversity stress. Therefore, it is significant to research AsA. GalUR gene is a key enzyme in conversion pathway of uronic acids which is a possible pathway about AsA biosynthesis. We hope we can provide theoretic reference for the pathway through our study.In this study, we cloned GalUR gene from Actinidia deliciosa cv.‘Qinmei’, and constructed three express vectors of the gene, which lay the foundation for lucubrating the function of GalUR gene. The main results in the study were as follows:1.Obtaining cDNA overall length of GalUR gene from kiwi fruit. The cDNA sequence length is 1028 bp , having an whole open reading frame of 990 bp, encoding 329 amino acid. It has a homology of the nucleotide sequences of 100% with Actinidia deliciosa (FG475088.1) through BLAST in Genebank, while the homology of amino acid sequences is above 70% with other plants.2.Constructing plant excessive express vector pWR/GalUR. Then we transformed it into EHA105 (Agrobacterium tumefacien) competent cells successfully.3.Constructing plant RNAi express vector pHGRV/GalUR and transforming it into EHA105 competent cells.4.We tested several indicators relating to AsA content and observed the changes of GalUR gene expression level with quantitative real-time RT-PCR in terms of different growth phases of kiwi fruits and different tissues of kiwi. Firstly, 30d after anthesis, both AsA content and the gene expression level in kiwi fruit were the highest, and then they maintained the certain levels. Secondly, in mature leaf, AsA content and the gene expression level were both higher than other tissues. The results indicated the expression of GalUR gene has close relation with AsA biosynthesis.5.Under the basis of restriction enzyme digestion, we built prokaryotic expression vector pET/GalUR and transformed into E.coli BL21 competent cells. After being induced by IPTG, an approximately 57kD specific protein band was obtained. According to the optimal expression conditions, polyclonal antibody was prepared.6.The total proteins of different tissues in kiwi extracted were used to be western blot. In the process, polyclonal antibody prepared was primary one. The protein expression level in mature leaf was the highest, which corresponded with the result of quantitative real-time RT-PCR. The polyclonal antibody could be distinguished specifically by antihelion in kiwi.更多还原