本文關鍵詞：高粱SbLIM1對木質素合成的轉錄調控及互作蛋白識別研究　出處：《山東大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 木質素 SbLIM1 凝膠阻滯 蛋白互作 酵母菌雙雜交

【摘要】：能源危機是世界各國均面臨的重要問題,生物能源特別是纖維素乙醇產業(yè)是最有潛力的替代能源解決途徑之一,降低木質素含量對提高纖維素的降解效率密切相關。研究高粱木質素代謝調控機理有望獲得低木質素轉基因能源植物,為纖維素乙醇產業(yè)提供優(yōu)質原料,對提高生產工藝水平,降低生產成本,提高經(jīng)濟效益,緩解能源緊張的局面發(fā)揮巨大作用。本實驗室前期構建13種高粱bmr突變體與野生型的SSH文庫,并通過芯片雜交篩選出153個差異表達基因,其中兩種轉錄因子基因SbLIM1、SbbHLH1在擬南芥中過表達,木質素含量明顯下降,木質素合成途徑中的部分基因及多個MYB轉錄因子的表達也出現(xiàn)不同程度的變化。通過構建PAL-box-min35S-GUS pStart載體35S-SbLIM1表達載體共同轉化洋蔥表皮(基因槍法),證實高粱的SbLIM1具有反式轉錄激活活性。利用蛋白互作軟件預測到多個SbLIM1的互作蛋白。通過酵母雙雜實驗,發(fā)現(xiàn)三種蛋白中有一種蛋白(Sb2276s002020.1)有較強的互作。親和吸附并純化大腸桿菌中表達的SbLIM1蛋白與高粱葉片總蛋白進行Pull-Down,洗脫其結合的蛋白進行電泳分離,質譜鑒定,發(fā)現(xiàn)其中有木質素合成的,Sb02g024220(CAD)及Sb01g041770(Class Ⅲ peroxidase 39)。另外還有大量糖代謝、氨基酸代謝、信號傳導蛋白等有待于進一步驗證。通過PCR克隆SbLIM1啟動子含有E-box(CANNTG)的三個DNA基序,分別與SbbHLH1轉錄因子進行結合,進行凝膠阻滯電泳,結果發(fā)現(xiàn)有兩個含有E-box的區(qū)域(E-box1和E-box2)發(fā)生電泳速度減緩,說明SbbHLH1轉錄因子最少可以和SbLIM1啟動子的兩個區(qū)域結合,這也預示著高粱SbLIM1的轉錄可能受到SbbHLH1的調控,即SbbHLH1可能位于SbLIM1的上游。將SbLIM1與SbbHLH1過表達擬南芥雜交,得到大量F1雜種,對其多個雜種進行生長發(fā)育、木質素含量測定,發(fā)現(xiàn)其木質素的含量比對照親本明顯下降,測定了多個木質素合成相關基因及轉錄因子的表達,也得到許多類似雙親且大幅下調表達的基因(例如At4CL,AtPAL1,AtCOMT)。說明兩個基因產物可能共同調節(jié)這些基因的轉錄,但是具體的分子機制還需進一步深入研究。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S514;Q943.2

【相似文獻】

相關碩士學位論文 前2條

1 李彤;高粱SbLIM1對木質素合成的轉錄調控及互作蛋白識別研究[D];山東大學;2017年

2 康亞麗;能源高粱幾種重要功能基因（SbHLH1，，SbLIM1）的研究[D];山東大學;2012年

本文編號：1345751