高粱SbLIM1对木质素合成的转录调控及互作蛋白识别研究

发布时间：2017-12-28 11:15

本文关键词：高粱SbLIM1对木质素合成的转录调控及互作蛋白识别研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：能源危机是世界各国均面临的重要问题,生物能源特别是纤维素乙醇产业是最有潜力的替代能源解决途径之一,降低木质素含量对提高纤维素的降解效率密切相关。研究高粱木质素代谢调控机理有望获得低木质素转基因能源植物,为纤维素乙醇产业提供优质原料,对提高生产工艺水平,降低生产成本,提高经济效益,缓解能源紧张的局面发挥巨大作用。本实验室前期构建13种高粱bmr突变体与野生型的SSH文库,并通过芯片杂交筛选出153个差异表达基因,其中两种转录因子基因SbLIM1、SbbHLH1在拟南芥中过表达,木质素含量明显下降,木质素合成途径中的部分基因及多个MYB转录因子的表达也出现不同程度的变化。通过构建PAL-box-min35S-GUS pStart载体35S-SbLIM1表达载体共同转化洋葱表皮(基因枪法),证实高粱的SbLIM1具有反式转录激活活性。利用蛋白互作软件预测到多个SbLIM1的互作蛋白。通过酵母双杂实验,发现三种蛋白中有一种蛋白(Sb2276s002020.1)有较强的互作。亲和吸附并纯化大肠杆菌中表达的SbLIM1蛋白与高粱叶片总蛋白进行Pull-Down,洗脱其结合的蛋白进行电泳分离,质谱鉴定,发现其中有木质素合成的,Sb02g024220(CAD)及Sb01g041770(Class Ⅲ peroxidase 39)。另外还有大量糖代谢、氨基酸代谢、信号传导蛋白等有待于进一步验证。通过PCR克隆SbLIM1启动子含有E-box(CANNTG)的三个DNA基序,分别与SbbHLH1转录因子进行结合,进行凝胶阻滞电泳,结果发现有两个含有E-box的区域(E-box1和E-box2)发生电泳速度减缓,说明SbbHLH1转录因子最少可以和SbLIM1启动子的两个区域结合,这也预示着高粱SbLIM1的转录可能受到SbbHLH1的调控,即SbbHLH1可能位于SbLIM1的上游。将SbLIM1与SbbHLH1过表达拟南芥杂交,得到大量F1杂种,对其多个杂种进行生长发育、木质素含量测定,发现其木质素的含量比对照亲本明显下降,测定了多个木质素合成相关基因及转录因子的表达,也得到许多类似双亲且大幅下调表达的基因(例如At4CL,AtPAL1,AtCOMT)。说明两个基因产物可能共同调节这些基因的转录,但是具体的分子机制还需进一步深入研究。
[Abstract]:Energy crisis is an important problem faced by all countries in the world. Bio energy, especially cellulose and ethanol industry is one of the most potential alternative energy solutions. Reducing lignin content is closely related to improving the efficiency of cellulose degradation. Studying the regulation mechanism of Lignin Metabolism in sorghum is expected to obtain low lignin transgenic energy plants, provide high-quality raw materials for the cellulose ethanol industry, and improve the production technology level, reduce production costs, increase economic benefits and ease energy shortage. SSH Library in our laboratory previously constructed 13 kinds of sorghum BMR mutant and wild type, and through hybridization selected 153 differentially expressed genes, of which two kinds of transcription factor gene SbLIM1, over expression of SbbHLH1 in Arabidopsis, lignin content decreased significantly, the expression of some genes in lignin biosynthesis and multiple MYB transcription factor there are different degrees of change. Through construction of PAL-box-min35S-GUS pStart vector 35S-SbLIM1 expression vector, the transformation of onion epidermis (gene gun method) confirmed that SbLIM1 of sorghum has trans transcriptional activation activity. The protein interaction software was used to predict the interaction proteins of multiple SbLIM1. The yeast two heterozygosity experiments showed that one of the three proteins (Sb2276s002020.1) had strong interaction. Affinity adsorption and purification of SbLIM1 protein expressed in Escherichia coli and total protein of sorghum leaves were carried out by Pull-Down. The proteins bound to them were separated by electrophoresis. Mass spectrometry identified that Sb02g024220 (CAD) and Sb01g041770 (Class III peroxidase 39) were synthesized from lignin. In addition, a large number of glucose metabolism, amino acid metabolism, signal transduction protein and so on need to be further verified. Through the PCR SbLIM1 promoter containing E-box (CANNTG) three DNA motifs, respectively with SbbHLH1 transcription factor, by gel retardation electrophoresis, we found two region with E-box (E-box1 and E-box2) electrophoresis slowed down, indicating that SbbHLH1 transcription factors can at least two promoter region and SbLIM1 the combination, which also indicates that the transcriptional regulation of sorghum SbLIM1 may be SbbHLH1, or SbbHLH1 SbLIM1 may be located upstream. SbLIM1 and SbbHLH1 overexpression hybridization, get a lot of F1 hybrids, the hybrids were developed, the content of lignin growth, found that the lignin content decreased significantly than the control parents, expression of a number of lignin synthesis related genes and transcription factors were also many similar parents and significantly downregulated the gene (such as At4CL, AtPAL1, AtCOMT). It is suggested that the two gene products may jointly regulate the transcription of these genes, but the specific molecular mechanisms need to be further studied.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S514;Q943.2

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