GmCHS基因生物信息学分析及过表达载体构建

发布时间：2018-04-22 04:21

本文选题：大豆胞囊线虫 + 黄酮　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：大豆胞囊线虫病是重要的土传大豆病害,严重影响大豆产量。目前种植抗性品种是重要的防治方式之一。抗性品种的选育依赖于新的抗性基因的发掘,因此发掘、克隆并鉴定新的抗性基因十分重要。黄酮在抗逆方面发挥着重要功能,与大豆对胞囊线虫的抗性相关,查尔酮合酶作为类黄酮合成途径的关键酶,可能通过类黄酮产物影响抗线虫的性状,具有较高的研究价值。本文通过研究前人的转录组数据发现大豆查尔酮合酶(Glycine max Chalcone synthase,GmCHS)基因在线虫侵染后的差异表达,克隆了 GmCHS基因CDS区序列,预测GmCHS蛋白为定位在真核细胞质内的以a螺旋与卷曲为主的中性蛋白,无跨膜结构且不存在信号肽。通过对GmCHS蛋白的互作研究,发现其与黄烷酮醇还原酶、黄烷酮3-羟化酶和黄酮醇合成酶家族交叉共表达,同时与查尔酮异构酶存在互作。对于互作网络进行GO注释的富集发现该网络响应刺激后,通过氧化还原反应调控黄酮类化合物代谢,参与抗性反应。因GmCHS在该代谢网络中处在核心位置,是主要的限速酶,从侧面证明该基因在抗性反应中的重要地位。为了更好的研究GmCHs基因是否参与大豆抗线虫的过程,构建了一个植物双元表达载体pCAMBIA1303::GmCHS,该质粒含有潮霉素抗性报告基因,GmCHS基因受35s启动子启动可在双子叶植物细胞内表达;在原核细胞内则表达卡那霉素抗性,可以高效的筛选克隆子。实验构建的载体可以为后续大豆转化实验提供材料,推动抗线基因发掘及大豆育种工作的进行。
[Abstract]:Soybean cyst nematode disease is an important soil-borne soybean disease, which seriously affects soybean yield. At present, planting resistant varieties is one of the most important control methods. The breeding of resistant varieties depends on the discovery of new resistance genes, so it is very important to discover, clone and identify new resistance genes. Flavonoids play an important role in stress resistance and are related to the resistance of soybean to cyst nematode. As the key enzyme of flavonoid synthesis pathway, chalcone synthase may influence the characters of nematode resistance through flavonoid products, so it has high research value. Based on previous transcriptome data, the differential expression of the gene of Glycine max Chalcone synthase (GmCHS) gene in soybean was found, and the CDS region of GmCHS gene was cloned. GmCHS protein was predicted to be a helix and crimp neutral protein located in eukaryotic cytoplasm without transmembrane structure and no signal peptide. The interaction of GmCHS protein with flavanone reductase, flavanone 3-hydroxylase and flavonol synthase family was found, and there was interaction with chalcone isomerase. After the interaction network was enriched with go annotation it was found that the network responded to the stimuli and regulated the metabolism of flavonoids through redox reaction and participated in the resistance response. Because GmCHS is at the core of the metabolic network, it is the main rate-limiting enzyme, which proves that the gene plays an important role in the resistance response. In order to better study whether GmCHs gene is involved in nematode resistance in soybean, a plant bivariate expression vector pCAMBIA13: 03: GmCHS was constructed. The plasmid contains hygromycin resistance reporter gene, GmCHS, which can be expressed in dicotyledonous plant cells by 35s promoter. The expression of kanamycin resistance in prokaryotic cells can effectively screen clones. The constructed vector can provide materials for the subsequent soybean transformation experiment and promote the discovery of resistance line genes and soybean breeding.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.651

【相似文献】