發(fā)布時間：2018-05-08 14:45

  本文選題：番茄 + SNAC4-9蛋白��； 參考：《天津大學(xué)》2016年碩士論文

【摘要】：NAC轉(zhuǎn)錄因子是植物特有的一類轉(zhuǎn)錄因子,目前在其生物學(xué)功能和調(diào)控網(wǎng)絡(luò)方面的研究已取得很大的進(jìn)展,從番茄中分離NAC蛋白并研究其調(diào)控的靶基因具有重要意義。課題組前期從番茄中分離出74個NAC轉(zhuǎn)錄因子,從中篩選出的SNAC4-9基因與番茄的生長發(fā)育、成熟衰老密切相關(guān)。因此本研究通過構(gòu)建原核表達(dá)載體以大量表達(dá)SNAC4-9蛋白,純化后進(jìn)一步驗證番茄NAC轉(zhuǎn)錄因子與乙烯合成關(guān)鍵基因間的相互作用。本文主要研究結(jié)果如下:1.對番茄SNAC4-9蛋白的理化性質(zhì)進(jìn)行預(yù)測后發(fā)現(xiàn),SNAC4/5/6/9為堿性蛋白質(zhì),SNAC7/8為中性蛋白質(zhì),且SNAC4-9均為親水性蛋白。對SNAC4-9蛋白啟動子區(qū)域的順式作用元件進(jìn)行分析,結(jié)果發(fā)現(xiàn)其啟動子區(qū)域不僅含有常見的TATAbox和CAATbox等基本轉(zhuǎn)錄元件,同時含有多種逆境應(yīng)答相關(guān)的順式作用元件如鹽脅迫應(yīng)答元件,低溫應(yīng)答元件和干旱應(yīng)答元件等,說明SNAC4-9轉(zhuǎn)錄因子可能參與番茄的生物及非生物脅迫應(yīng)答過程。同時含有激素響應(yīng)元件如乙烯應(yīng)答元件,ABA響應(yīng)元件和赤霉素響應(yīng)元件,說明SNAC4-9蛋白可能受相應(yīng)激素的誘導(dǎo)表達(dá)。2.成功構(gòu)建原核表達(dá)載體pET-SNAC4/5/7/8/9,利用IPTG誘導(dǎo)表達(dá)重組蛋白后發(fā)現(xiàn),SNAC4/7/8/9主要以包涵體蛋白的形式存在,SNAC5則主要以可溶性蛋白的形式存在。在優(yōu)化SNAC4-9蛋白的表達(dá)方案后,結(jié)果發(fā)現(xiàn)降低表達(dá)溫度和IPTG終濃度,延長表達(dá)時間以增加可溶性蛋白表達(dá)量的方案僅適用于目的蛋白以可溶性蛋白存在的情況。3.選擇最優(yōu)方案表達(dá)SNAC4/9蛋白,利用Ni柱親和層析純化SNAC4/9蛋白,同時采用切膠純化目的蛋白的方案純化SNAC4/9蛋白,然后對純化的SNAC4/9蛋白進(jìn)行復(fù)性,結(jié)果表明兩種方案均獲得了純度較高的目的蛋白。4.通過凝膠阻滯實驗在番茄果實體外確定SNAC4/9轉(zhuǎn)錄因子調(diào)控的目的基因。結(jié)果表明SNAC4轉(zhuǎn)錄因子可與LeACS2/LeACS4啟動子結(jié)合,而SNAC9轉(zhuǎn)錄因子可與LeACO1/LeACO4/LeACS2/LeACS4的啟動子結(jié)合,初步證實SNAC4可直接調(diào)控LeACS2/LeACS4,SNAC9可直接調(diào)控LeACO1/LeACO4/LeACS2/LeACS4,SNAC4/9轉(zhuǎn)錄因子通過調(diào)控果實的乙烯生物合成過程而影響番茄果實的成熟。
[Abstract]:NAC transcription factor is a kind of plant specific transcription factor. At present, great progress has been made in the study of its biological function and regulatory network. It is of great significance to isolate NAC protein from tomato and study its target gene. 74 NAC transcription factors were isolated from tomato in early stage. The SNAC4-9 gene was closely related to tomato growth and maturation and senescence. In this study, the prokaryotic expression vector was constructed to express SNAC4-9 protein in large quantities, and the interaction between tomato NAC transcription factors and key genes of ethylene synthesis was further verified after purification. The main results of this paper are as follows: 1: 1. The physicochemical properties of tomato SNAC4-9 protein were predicted. It was found that SNAC4 / 5 / 6 / 9 was a basic protein, SNAC7 / 8 was neutral protein, and SNAC4-9 was hydrophilic protein. The cis-acting elements in the promoter region of SNAC4-9 protein were analyzed. The results showed that the promoter region contained not only basic transcription elements such as TATAbox and CAATbox, but also a variety of cis-acting elements related to stress response, such as salt stress response elements. The low temperature response element and drought response element indicate that SNAC4-9 transcription factors may be involved in the biotic and abiotic stress response process of tomato. It also contains hormone response elements such as ethylene response element and gibberellin response element, indicating that SNAC4-9 protein may be induced by hormone expression. 2. The prokaryotic expression vector pET-SNAC4 / 5 / 7 / 8 / 9 was successfully constructed. After IPTG was used to induce the expression of recombinant protein, it was found that SNAC4 / 7 / 8 / 9 existed mainly in the form of inclusion body protein, while SNAC5 existed mainly in the form of soluble protein. After optimizing the expression scheme of SNAC4-9 protein, it was found that the scheme of decreasing the expression temperature and the final concentration of IPTG, prolonging the expression time to increase the expression of soluble protein was only suitable for the situation where the target protein existed as soluble protein. SNAC4/9 protein was expressed by the best method, SNAC4/9 protein was purified by Ni column affinity chromatography, and SNAC4/9 protein was purified by the method of digesting the target protein. Then the purified SNAC4/9 protein was renatured. The results showed that the target protein with high purity was obtained by both methods. The aim gene of SNAC4/9 transcription factor regulation was identified in tomato fruit by gel retardation in vitro. The results showed that SNAC4 transcription factors could bind to LeACS2/LeACS4 promoter, while SNAC9 transcription factors could bind to LeACO1/LeACO4/LeACS2/LeACS4 promoter. It was preliminarily confirmed that SNAC4 can directly regulate LeACS _ 2 / LeACS _ 4 / SNAC9 and directly regulate the ripening of tomato fruit by regulating the transcription factors of LeACS _ 2 / LeACS _ 2 / LeACS _ 4 / SNAC _ 4 / 9 by regulating the ethylene biosynthesis process of tomato fruit.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

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本文編號：1861813