苹果MdHB1基因启动子的克隆及特征分析
发布时间：2019-04-27 15:14
【摘要】：MdHB1是一个转录因子,属于苹果HD-Zip I亚家族的成员,根据先前的报道,MdHB1能够与MdACO1启动子结合,进而调控乙烯生物合成。本试验以‘皇家嘎啦’叶片为试材,克隆MdHB1基因启动子,然后以烟草为材料,利用农杆菌介导的瞬时转化技术来探究苹果MdHB1基因启动子的特性。本试验结果如下:1、克隆了长度为1057 bp的MdHB1基因启动子,利用生物信息学软件对MdHB1基因启动子进行转录起始位点和顺式作用元件的分析,结果显示转录起始位点(TSS)、CAAT BOX、TATA BOX分别位于翻译起始位点(ATG)上游266 bp、319 bp、308 bp的位置。在这一段基因启动子区域内存在响应激素和环境因素的顺式作用元件,如ERE、ABRE、ARE、G-Box等。2、启动子5’端序列缺失试验表明,当启动子长度为680 bp时,活性最低,当启动子长度为266 bp时,仍具有较高的启动子活性。5'UTR(非编码区域)在基因表达过程中可能起到一个积极的作用。3、通过农杆菌介导的瞬时转化技术,获得含有长度为1057 bp启动子的转基因烟草,在生物和非生物因素的处理下,启动子受到乙烯、黑暗的正调控作用,而脱落酸(ABA)、水杨酸(SA)、假单胞丁香杆菌(DC3000)和机械伤对启动子的正调控作用较微弱,赤霉素(GA)则抑制启动子的活性。4、乙烯响应元件(距ATG上游576 bp)能够响应外源乙烯正调控作用,当外源乙烯处理浓度逐渐增大时,正调控效应逐渐升高达到最大值,然后逐渐降低,500 mg L-1乙烯利能最大程度的提升乙烯响应元件活性。
[Abstract]:MdHB1 is a transcription factor belonging to the apple HD-Zip I subfamily. According to previous reports, MdHB1 binds to the MdACO1 promoter, which in turn regulates ethylene biosynthesis. The MdHB1 gene promoter was cloned from the leaves of 'Royal Gala', and then the characteristics of apple MdHB1 gene promoter were investigated by Agrobacterium-mediated transient transformation technique. The results are as follows: 1. The promoter of MdHB1 gene with the length of 1057 bp was cloned. The transcriptional initiation sites and cis-acting elements of the promoter of MdHB1 gene were analyzed by bioinformatics software. The results showed that the transcription initiation site of MdHB1 gene promoter was (TSS),. CAAT BOX,TATA BOX was located at 266 bp,319 bp,308 bp upstream of the translation initiation site (ATG). In the promoter region of this gene, there are cis-acting elements that respond to hormone and environmental factors, such as ERE,ABRE,ARE,G-Box et al., the deletion test of the 5 'end of the promoter shows that when the promoter length is 680bp, the activity of the promoter is the lowest. 5'UTR (non-coding region) may play an active role in gene expression. 3, transient transformation technology mediated by Agrobacterium tumefaciens, 5'UTR (non-coding region) may play an active role in gene expression. 3. When promoter length is 266bp, 5'UTR (non-coding region) may play an active role in gene expression. Transgenic tobacco with a length of 1057 bp promoter was obtained. Under the treatment of biological and abiotic factors, the promoter was positively regulated by ethylene and dark, while the abscisic acid (ABA), salicylic acid (SA), was obtained. The positive regulation of promoter by DC3000 and mechanical injury was weak, while gibberellin (GA) inhibited the activity of promoter. 4. Ethylene response element (576 bp upstream of ATG) could respond to the positive regulation of exogenous ethylene. When the concentration of exogenous ethylene increased gradually, the positive regulation effect increased to the maximum value, then decreased gradually, and the ethylene response element activity of 500 mg / L 路L ~ (- 1) ethephon increased to the maximum extent.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S661.1
[Abstract]:MdHB1 is a transcription factor belonging to the apple HD-Zip I subfamily. According to previous reports, MdHB1 binds to the MdACO1 promoter, which in turn regulates ethylene biosynthesis. The MdHB1 gene promoter was cloned from the leaves of 'Royal Gala', and then the characteristics of apple MdHB1 gene promoter were investigated by Agrobacterium-mediated transient transformation technique. The results are as follows: 1. The promoter of MdHB1 gene with the length of 1057 bp was cloned. The transcriptional initiation sites and cis-acting elements of the promoter of MdHB1 gene were analyzed by bioinformatics software. The results showed that the transcription initiation site of MdHB1 gene promoter was (TSS),. CAAT BOX,TATA BOX was located at 266 bp,319 bp,308 bp upstream of the translation initiation site (ATG). In the promoter region of this gene, there are cis-acting elements that respond to hormone and environmental factors, such as ERE,ABRE,ARE,G-Box et al., the deletion test of the 5 'end of the promoter shows that when the promoter length is 680bp, the activity of the promoter is the lowest. 5'UTR (non-coding region) may play an active role in gene expression. 3, transient transformation technology mediated by Agrobacterium tumefaciens, 5'UTR (non-coding region) may play an active role in gene expression. 3. When promoter length is 266bp, 5'UTR (non-coding region) may play an active role in gene expression. Transgenic tobacco with a length of 1057 bp promoter was obtained. Under the treatment of biological and abiotic factors, the promoter was positively regulated by ethylene and dark, while the abscisic acid (ABA), salicylic acid (SA), was obtained. The positive regulation of promoter by DC3000 and mechanical injury was weak, while gibberellin (GA) inhibited the activity of promoter. 4. Ethylene response element (576 bp upstream of ATG) could respond to the positive regulation of exogenous ethylene. When the concentration of exogenous ethylene increased gradually, the positive regulation effect increased to the maximum value, then decreased gradually, and the ethylene response element activity of 500 mg / L 路L ~ (- 1) ethephon increased to the maximum extent.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S661.1
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