本文關(guān)鍵詞：高粱SbLIM1對(duì)木質(zhì)素合成的轉(zhuǎn)錄調(diào)控及互作蛋白識(shí)別研究　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 木質(zhì)素 SbLIM1 凝膠阻滯 蛋白互作 酵母菌雙雜交

【摘要】：能源危機(jī)是世界各國(guó)均面臨的重要問(wèn)題,生物能源特別是纖維素乙醇產(chǎn)業(yè)是最有潛力的替代能源解決途徑之一,降低木質(zhì)素含量對(duì)提高纖維素的降解效率密切相關(guān)。研究高粱木質(zhì)素代謝調(diào)控機(jī)理有望獲得低木質(zhì)素轉(zhuǎn)基因能源植物,為纖維素乙醇產(chǎn)業(yè)提供優(yōu)質(zhì)原料,對(duì)提高生產(chǎn)工藝水平,降低生產(chǎn)成本,提高經(jīng)濟(jì)效益,緩解能源緊張的局面發(fā)揮巨大作用。本實(shí)驗(yàn)室前期構(gòu)建13種高粱bmr突變體與野生型的SSH文庫(kù),并通過(guò)芯片雜交篩選出153個(gè)差異表達(dá)基因,其中兩種轉(zhuǎn)錄因子基因SbLIM1、SbbHLH1在擬南芥中過(guò)表達(dá),木質(zhì)素含量明顯下降,木質(zhì)素合成途徑中的部分基因及多個(gè)MYB轉(zhuǎn)錄因子的表達(dá)也出現(xiàn)不同程度的變化。通過(guò)構(gòu)建PAL-box-min35S-GUS pStart載體35S-SbLIM1表達(dá)載體共同轉(zhuǎn)化洋蔥表皮(基因槍法),證實(shí)高粱的SbLIM1具有反式轉(zhuǎn)錄激活活性。利用蛋白互作軟件預(yù)測(cè)到多個(gè)SbLIM1的互作蛋白。通過(guò)酵母雙雜實(shí)驗(yàn),發(fā)現(xiàn)三種蛋白中有一種蛋白(Sb2276s002020.1)有較強(qiáng)的互作。親和吸附并純化大腸桿菌中表達(dá)的SbLIM1蛋白與高粱葉片總蛋白進(jìn)行Pull-Down,洗脫其結(jié)合的蛋白進(jìn)行電泳分離,質(zhì)譜鑒定,發(fā)現(xiàn)其中有木質(zhì)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(hào)傳導(dǎo)蛋白等有待于進(jìn)一步驗(yàn)證。通過(guò)PCR克隆SbLIM1啟動(dòng)子含有E-box(CANNTG)的三個(gè)DNA基序,分別與SbbHLH1轉(zhuǎn)錄因子進(jìn)行結(jié)合,進(jìn)行凝膠阻滯電泳,結(jié)果發(fā)現(xiàn)有兩個(gè)含有E-box的區(qū)域(E-box1和E-box2)發(fā)生電泳速度減緩,說(shuō)明SbbHLH1轉(zhuǎn)錄因子最少可以和SbLIM1啟動(dòng)子的兩個(gè)區(qū)域結(jié)合,這也預(yù)示著高粱SbLIM1的轉(zhuǎn)錄可能受到SbbHLH1的調(diào)控,即SbbHLH1可能位于SbLIM1的上游。將SbLIM1與SbbHLH1過(guò)表達(dá)擬南芥雜交,得到大量F1雜種,對(duì)其多個(gè)雜種進(jìn)行生長(zhǎng)發(fā)育、木質(zhì)素含量測(cè)定,發(fā)現(xiàn)其木質(zhì)素的含量比對(duì)照親本明顯下降,測(cè)定了多個(gè)木質(zhì)素合成相關(guān)基因及轉(zhuǎn)錄因子的表達(dá),也得到許多類似雙親且大幅下調(diào)表達(dá)的基因(例如At4CL,AtPAL1,AtCOMT)。說(shuō)明兩個(gè)基因產(chǎn)物可能共同調(diào)節(jié)這些基因的轉(zhuǎn)錄,但是具體的分子機(jī)制還需進(jìn)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S514;Q943.2

【相似文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 李彤;高粱SbLIM1對(duì)木質(zhì)素合成的轉(zhuǎn)錄調(diào)控及互作蛋白識(shí)別研究[D];山東大學(xué);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(xué);2012年

本文編號(hào)：1345751