本文關(guān)鍵詞： 棉花 谷胱甘肽轉(zhuǎn)移酶 蛋白質(zhì)印跡法 生物信息學(xué) 鹽脅迫　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：土地鹽堿化作為一種非生物脅迫,已成為制約農(nóng)業(yè)生產(chǎn)發(fā)展的重要限制因素。棉花作為世界上重要的經(jīng)濟(jì)作物,對(duì)高溫、干旱、高鹽等非生物脅迫具有一定耐性,但鹽濃度過高會(huì)造成棉花滲透脅迫和離子毒害,導(dǎo)致棉花產(chǎn)量和質(zhì)量的下降。谷胱甘肽轉(zhuǎn)移酶(Glutathione transferases,GSTs,EC 2.5.1.18)是一類多功能蛋白家族,在植物體抵御外源物質(zhì)毒害及抵抗多種非生物脅迫中起著重要作用。目前,對(duì)GST在耐鹽方面的研究多集中在煙草、擬南芥等模式生物中,但是對(duì)棉花中GST的耐鹽機(jī)理研究還鮮有報(bào)道。本研究首先利用生物信息學(xué)方法,對(duì)棉花谷胱甘肽轉(zhuǎn)移酶理化性質(zhì)、二級(jí)結(jié)構(gòu)、信號(hào)肽及亞細(xì)胞定位、三維結(jié)構(gòu)進(jìn)行預(yù)測(cè),對(duì)氨基酸序列進(jìn)行了同源性比對(duì)并構(gòu)建系統(tǒng)進(jìn)化樹。在此基礎(chǔ)上以中棉所79為實(shí)驗(yàn)材料,利用NaCl溶液模擬鹽脅迫對(duì)棉花葉片中GST的表達(dá)進(jìn)行分析。對(duì)基于SDS-PAGE的western blot檢測(cè)方法進(jìn)行了優(yōu)化,確定了適合western blot的最佳蛋白樣品制備方法和電泳上樣量,對(duì)鹽脅迫下棉花GST的表達(dá)變化進(jìn)行了分析。在此基礎(chǔ)上,建立了基于雙向電泳的western blot分析方法,得到了鹽脅迫下棉花GST的差異表達(dá)圖譜,并對(duì)western blot檢測(cè)出的GST差異蛋白點(diǎn)進(jìn)行量化分析,獲得棉花GST亞型響應(yīng)鹽脅迫的表達(dá)情況。主要研究結(jié)果如下:1、對(duì)棉花中谷胱甘肽轉(zhuǎn)移酶進(jìn)行生物信息學(xué)分析。利用多種工具在理化性質(zhì)、二級(jí)結(jié)構(gòu)、亞細(xì)胞定位、親水性/疏水性方面進(jìn)行預(yù)測(cè)。結(jié)果顯示GST含有225個(gè)氨基酸,分子量為26KDa,等電點(diǎn)為5.75,屬于親水性蛋白質(zhì),蛋白性質(zhì)穩(wěn)定為細(xì)胞質(zhì)蛋白。利用Blastp進(jìn)行同原序列比對(duì),選擇6條氨基酸序列構(gòu)建系統(tǒng)進(jìn)化樹。2、優(yōu)化了棉花葉片蛋白的制備方法并確定適用于western blot的上樣量。在參考本實(shí)驗(yàn)室已有的提取方法及查找文獻(xiàn)的基礎(chǔ)上,篩選適合于1D-western blot檢測(cè)GST的最適樣品制備方法,最終確定了提取蛋白粉溶解上樣的方法。通過不同上樣量的western blot顯影效果,發(fā)現(xiàn)GST適合于較高的上樣量。3、利用SDS-PAGE和western blot方法對(duì)鹽脅迫下的棉花谷胱甘肽轉(zhuǎn)移酶表達(dá)量的變化進(jìn)行分析。在鹽濃度相同的條件下,不同的脅迫時(shí)間造成棉花葉片中GST的差異表達(dá)。結(jié)果顯示,對(duì)照組棉花葉片內(nèi)GST表達(dá)穩(wěn)定,鹽脅迫12小時(shí)后GST的表達(dá)受到抑制,處理24小時(shí)GST的表達(dá)量開始上升,并隨著時(shí)間的增長(zhǎng)而不斷上升。4、建立了基于雙向電泳技術(shù)的western blot檢測(cè)體系。對(duì)脅迫24h的棉花幼苗葉片蛋白進(jìn)行雙向電泳分析,獲得棉花葉片響應(yīng)鹽脅迫的差異蛋白圖譜。在此基礎(chǔ)上對(duì)蛋白凝膠經(jīng)行western blot分析得到2D-western blot的差異蛋白點(diǎn),利用ImageMaster 2D PLatinum7軟件對(duì)不同的GST蛋白點(diǎn)進(jìn)行匹配和量化分析。結(jié)果顯示,棉花中共有8個(gè)GST蛋白點(diǎn)響應(yīng)鹽脅迫,并出現(xiàn)差異表達(dá),其中2個(gè)點(diǎn)表達(dá)上調(diào),6個(gè)點(diǎn)表達(dá)下調(diào)。這表明,棉花葉片中存在多種亞型的谷胱甘肽轉(zhuǎn)移酶,且在鹽脅迫處理后出現(xiàn)不同的表達(dá)情況,這一結(jié)果為后續(xù)針對(duì)谷胱甘肽轉(zhuǎn)移酶亞型在耐鹽機(jī)制中的作用及基因改良育種方面的研究打下基礎(chǔ)。
[Abstract]:Land salinization as an abiotic stress, has become an important restriction factor for the development of agricultural production. Cotton as the most important economic crops in the world, the high temperature, drought, high salt and other abiotic stress has certain tolerance, but the salt concentration is too high will cause osmotic stress and ion toxicity of cotton, resulting in decreased yield and quality cotton. Glutathione S-transferase (Glutathione, transferases, GSTs, EC, 2.5.1.18) is a multifunctional protein family, resist exogenous substances and a variety of non poison resistance plays an important role in abiotic stress in plants. At present, the research of GST in salt area concentrated in tobacco, Arabidopsis and other model organisms. But the cotton GST in the mechanism of salt tolerance research is rarely reported. In this study, using bioinformatics methods, physical and chemical properties of cotton enzyme glutathione, two level structure, signal peptide and subcellular set A 3D structure prediction of the amino acid sequence homology and phylogenetic tree was constructed. On the basis of CCRI 79 as experimental materials, simulation of salt stress on the expression of GST in cotton leaves were analyzed by using NaCl solution. The Western blot detection method based on SDS-PAGE were optimized to determine the best protein samples preparation method and electrophoresis for Western blot sample, on expression of GST of cotton under salt stress were analyzed. On this basis, the two dimensional electrophoresis analysis of Western blot based on the difference, under salt stress of cotton GST expression profile, and the GST difference detection of Western blot protein. The quantitative analysis of cotton GST subtype expression in response to salt stress. The main results are as follows: 1, the glutathione S-transferase bioinformatics analysis using cotton. A tool in the physical and chemical properties, two level structure, subcellular localization, hydrophilic / hydrophobic side prediction. The results showed that GST containing 225 amino acids, molecular weight of 26KDa and isoelectric point of 5.75, is a hydrophilic protein, protein stability for cytoplasmic protein. Using Blastp in the same primary sequence, selection 6 amino acid sequence phylogenetic tree of.2 protein in cotton leaves, optimizing preparation method and determined for Western blot sample. Based on the existing laboratory reference extraction method and search literature, screening for 1D-western blot detection GST the optimum sample preparation methods, and ultimately determine the method extraction of protein powder dissolved sample. Through the Western blot imaging effect of different sample volume, sample volume of.3 found that GST is suitable for high, using SDS-PAGE and Western blot method under Salt Stress on cotton glutathiones Shift enzyme expression analysis. In the condition of the same concentration of salt under different stress time, differential expression in cotton leaves caused by GST. The results showed that the control group GST was stably expressed in cotton leaves, the expression of GST was inhibited by salt stress after 12 hours, the expression of 24 hours GST began to rise, and with the increase of time and the increasing of.4, established the Western blot detection system based on two-dimensional electrophoresis. For two-dimensional electrophoresis analysis of stress 24h of cotton seedling leaf protein, cotton leaf protein profile obtained in response to salt stress. On the basis of protein gel by Western blot analysis 2D-western blot proteins, matching and quantitative analysis of different GST protein using ImageMaster 2D PLatinum7 software. The results showed that a total of 8 cotton GST protein point in response to salt stress, and the difference The expression, of which 2 spots up-regulated and 6 spots were down regulated. This suggests that multiple isoforms of glutathione S-transferase in cotton leaves, and the expression in different salt stress treatment, the results lay the foundation for the further research on glutathione S-transferase isoforms in salt tolerance mechanism and genetically modified breeding.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S562

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