桃蔗糖转运蛋白基因的功能验证与PpSUT2蛋白表达分析

发布时间：2018-06-12 09:48

本文选题：桃 + 蔗糖转运蛋白　；参考：《河南农业大学》2016年硕士论文

【摘要】：桃(Prunus Persica(L.)Batsch.)因其营养丰富、风味独特在园艺产业中占据重要地位。桃果实的品质受多种因素影响,如光合产物的转化和积累。蔗糖作为碳水化合物的主要存在形式,在源叶合成后经过韧皮部的运输到达各库器官,如果实中。桃果实的品质和产量很大程度上取决于所含碳水化合物的种类和数量。蔗糖转运蛋白作为碳水化合物装载进韧皮部,且从合成部位运输到有需求组织部位的主要介质,负责蔗糖的装载、卸载和分配。通过研究蔗糖转运蛋白的作用机制,可以帮助理解决定果实生长和品质的蔗糖积累的过程。因此,对植物蔗糖转运蛋白的深入探索研究是很有必要的。该试验在本课题组的研究基础上,从桃中克隆出桃蔗糖转运蛋白家族的3个成员PpSUT1,PpSUT2和PpSUT4,利用缺陷型酵母对其进行功能验证,通过对PpSUT2进行原核表达,制备了多克隆抗体,并对桃各组织中PpSUT2的蛋白表达进行分析,为基因的免疫细胞定位、蛋白质互作因子等研究提供了重要依据。主要作了如下几项研究:1.桃SUTs的克隆和序列分析从桃中克隆出其蔗糖转运蛋白家族基因PpSUT1(KU230443),PpSUT2(KJ152144)和PpSUT4(KU198999)分别与桃基因组序列ppa004033m,ppa003041m和ppa004620m相一致。此处得到的PpSUT2基因含有长度为1830bp的ORF,比此前克隆的PpSUT2(1782bp)多出47个碱基,编码长度为609个氨基酸的蛋白质。2.桃SUTs在酵母中的功能分析通过构建酵母表达载体,将桃的3个SUT基因在具有蔗糖吸收缺陷的酵母菌株SUSY7/ura3中进行异源表达,PpSUT1、PpSUT2和PpSUT4在SD-蔗糖培养基上都表现出了功能的互补,表明这3个桃蔗糖运蛋白基因都分别编码一个具有蔗糖转运活性的功能性蛋白。3.桃PpSUT2的原核表达及抗体制备根据PpSUT2基因的ORF序列,构建p ET32a-PpSUT2重组质粒,在表达菌株大肠杆菌BL21(DE3)Plys S中经IPTG诱导表达,所得到融合蛋白的分子量约为84k D。以纯化的PpSUT2-His蛋白为抗原进行兔免疫,用间接ELISA对抗血清进行效价检测,其效价达到了1:12800,Western blot分析显示该抗体能特异性识别PpSUT2融合蛋白,表明制备的PpSUT2多克隆抗体具有较高的检测灵敏度和特异性。4.桃PpSUT2的蛋白组织特异性表达提取桃不同组织的总蛋白,进行Western blot检测,在分子量84kD左右处出现特异的蛋白质条带,证明所制备的抗体可以特异性识别桃PpSUT2蛋白,并且在桃的各个组织器官中都检测到了PpSUT2蛋白的表达,通过分析PpSUT2蛋白的表达模式,推测其功能是在果实发育期负责韧皮部蔗糖的转运和卸载,还有可能起到信号蛋白的功能。
[Abstract]:Prunus Persica L. Batsch. Because of its rich nutrition, unique flavor occupies an important position in horticultural industry. The quality of peach fruit is affected by many factors, such as the transformation and accumulation of photosynthetic products. Sucrose, as the main carbohydrate form, is transported through phloem to the storage organs after the source leaves are synthesized. The quality and yield of peach fruits depend to a great extent on the kinds and quantities of carbohydrates contained. Sucrose transporters are used as carbohydrates into the phloem and are transported from the synthetic site to the required tissue sites, which are responsible for the loading, unloading and distribution of sucrose. The study of the mechanism of sucrose transporter can help to understand the process of sucrose accumulation which determines the growth and quality of fruit. Therefore, it is necessary to explore the plant sucrose transporter. Three members of the peach sucrose transporter family, PpSUT1, PpSUT2 and PpSUT4, were cloned from peach on the basis of our research. The protein expression of PpSUT2 in peach tissues was analyzed, which provided an important basis for the study of immunocyte localization of genes and protein interaction factors. The following studies were carried out: 1. Cloning and sequence analysis of sucrose transporter family genes PpSUT1 (KU230443) and PpSUT4 (KJ152144) and PpSUT4 (KU198999) were cloned from peach, respectively, which were consistent with the genomic sequence ppa004033mppa003041m and ppa004620m, respectively. The obtained PpSUT2 gene contains an ORF with a length of 1830bp, which is 47 bases longer than the previously cloned PpSUT2Amino 1782bp) and encodes a 609 amino acid protein. Functional Analysis of Peach SUTs in Saccharomyces cerevisiae by constructing yeast expression Vectors, three sut genes of peach were expressed in sucrose deficient yeast strain SUSY7 / ura3. Both PpSUT1, PpSUT2 and PpSUT4 showed complementary functions on SD-sucrose medium. These three sucrose transporter genes all encode a functional protein. 3 with sucrose transport activity. Prokaryotic expression of PpSUT2 and preparation of PpSUT2 antibody the recombinant plasmid of pET32a-PpSUT2 was constructed according to the ORF sequence of PpSUT2 gene. The recombinant plasmid was induced by IPTG to express pET32a-PpSUT2. The molecular weight of the fusion protein was about 84kD. The purified PpSUT2-His protein was used as antigen to immunize rabbits. The titer of PpSUT2 fusion protein was detected by indirect Elisa. The titer of the purified PpSUT2-His protein reached 1: 12800m Western blot analysis showed that the antibody could specifically recognize PpSUT2 fusion protein. The results showed that the PpSUT2 polyclonal antibody had high detection sensitivity and specificity. 4. The protein specific expression of PpSUT2 was used to extract the total proteins from different peach tissues. Western blot was used to detect the specific protein bands at the molecular weight of 84kD. It was proved that the prepared antibody could specifically recognize the PpSUT2 protein of peach. The expression of PpSUT2 protein was detected in all tissues and organs of peach. By analyzing the expression pattern of PpSUT2 protein, it was inferred that the function of PpSUT2 protein was to transport and unload sucrose in phloem during fruit development, and it might also play the role of signal protein.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S662.1

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