【摘要】：MdHB1是一個轉(zhuǎn)錄因子,屬于蘋果HD-Zip I亞家族的成員,根據(jù)先前的報道,MdHB1能夠與MdACO1啟動子結(jié)合,進而調(diào)控乙烯生物合成。本試驗以‘皇家嘎啦’葉片為試材,克隆MdHB1基因啟動子,然后以煙草為材料,利用農(nóng)桿菌介導(dǎo)的瞬時轉(zhuǎn)化技術(shù)來探究蘋果MdHB1基因啟動子的特性。本試驗結(jié)果如下:1、克隆了長度為1057 bp的MdHB1基因啟動子,利用生物信息學(xué)軟件對MdHB1基因啟動子進行轉(zhuǎn)錄起始位點和順式作用元件的分析,結(jié)果顯示轉(zhuǎn)錄起始位點(TSS)、CAAT BOX、TATA BOX分別位于翻譯起始位點(ATG)上游266 bp、319 bp、308 bp的位置。在這一段基因啟動子區(qū)域內(nèi)存在響應(yīng)激素和環(huán)境因素的順式作用元件,如ERE、ABRE、ARE、G-Box等。2、啟動子5’端序列缺失試驗表明,當啟動子長度為680 bp時,活性最低,當啟動子長度為266 bp時,仍具有較高的啟動子活性。5'UTR(非編碼區(qū)域)在基因表達過程中可能起到一個積極的作用。3、通過農(nóng)桿菌介導(dǎo)的瞬時轉(zhuǎn)化技術(shù),獲得含有長度為1057 bp啟動子的轉(zhuǎn)基因煙草,在生物和非生物因素的處理下,啟動子受到乙烯、黑暗的正調(diào)控作用,而脫落酸(ABA)、水楊酸(SA)、假單胞丁香桿菌(DC3000)和機械傷對啟動子的正調(diào)控作用較微弱,赤霉素(GA)則抑制啟動子的活性。4、乙烯響應(yīng)元件(距ATG上游576 bp)能夠響應(yīng)外源乙烯正調(diào)控作用,當外源乙烯處理濃度逐漸增大時,正調(diào)控效應(yīng)逐漸升高達到最大值,然后逐漸降低,500 mg L-1乙烯利能最大程度的提升乙烯響應(yīng)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S661.1

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