番茄NAC转录因子蛋白的表达载体构建、纯化及靶基因研究

发布时间：2018-05-08 14:45

本文选题：番茄 + SNAC4-9蛋白　；参考：《天津大学》2016年硕士论文

【摘要】：NAC转录因子是植物特有的一类转录因子,目前在其生物学功能和调控网络方面的研究已取得很大的进展,从番茄中分离NAC蛋白并研究其调控的靶基因具有重要意义。课题组前期从番茄中分离出74个NAC转录因子,从中筛选出的SNAC4-9基因与番茄的生长发育、成熟衰老密切相关。因此本研究通过构建原核表达载体以大量表达SNAC4-9蛋白,纯化后进一步验证番茄NAC转录因子与乙烯合成关键基因间的相互作用。本文主要研究结果如下:1.对番茄SNAC4-9蛋白的理化性质进行预测后发现,SNAC4/5/6/9为碱性蛋白质,SNAC7/8为中性蛋白质,且SNAC4-9均为亲水性蛋白。对SNAC4-9蛋白启动子区域的顺式作用元件进行分析,结果发现其启动子区域不仅含有常见的TATAbox和CAATbox等基本转录元件,同时含有多种逆境应答相关的顺式作用元件如盐胁迫应答元件,低温应答元件和干旱应答元件等,说明SNAC4-9转录因子可能参与番茄的生物及非生物胁迫应答过程。同时含有激素响应元件如乙烯应答元件,ABA响应元件和赤霉素响应元件,说明SNAC4-9蛋白可能受相应激素的诱导表达。2.成功构建原核表达载体pET-SNAC4/5/7/8/9,利用IPTG诱导表达重组蛋白后发现,SNAC4/7/8/9主要以包涵体蛋白的形式存在,SNAC5则主要以可溶性蛋白的形式存在。在优化SNAC4-9蛋白的表达方案后,结果发现降低表达温度和IPTG终浓度,延长表达时间以增加可溶性蛋白表达量的方案仅适用于目的蛋白以可溶性蛋白存在的情况。3.选择最优方案表达SNAC4/9蛋白,利用Ni柱亲和层析纯化SNAC4/9蛋白,同时采用切胶纯化目的蛋白的方案纯化SNAC4/9蛋白,然后对纯化的SNAC4/9蛋白进行复性,结果表明两种方案均获得了纯度较高的目的蛋白。4.通过凝胶阻滞实验在番茄果实体外确定SNAC4/9转录因子调控的目的基因。结果表明SNAC4转录因子可与LeACS2/LeACS4启动子结合,而SNAC9转录因子可与LeACO1/LeACO4/LeACS2/LeACS4的启动子结合,初步证实SNAC4可直接调控LeACS2/LeACS4,SNAC9可直接调控LeACO1/LeACO4/LeACS2/LeACS4,SNAC4/9转录因子通过调控果实的乙烯生物合成过程而影响番茄果实的成熟。
[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.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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