本文關(guān)鍵詞：番茄果實(shí)中特異性表達(dá)的SlSWEET14基因啟動(dòng)子的克隆與功能分析　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 番茄 SlSWEET14 啟動(dòng)子 GUS 表達(dá)模式

【摘要】：SWEETs基因家族是新發(fā)現(xiàn)的膜轉(zhuǎn)運(yùn)蛋白,其可以參與蔗糖、果糖、葡萄糖等糖類的轉(zhuǎn)運(yùn)。前期研究表明番茄中SISWEET14為果實(shí)中特異性表達(dá)的基因,其參與轉(zhuǎn)運(yùn)糖類,但其具體作用方式尚不明確。在調(diào)控基因表達(dá)的過程中啟動(dòng)子起到關(guān)鍵調(diào)控作用,所以通過對(duì)基因啟動(dòng)子及其順式作用元件的生物學(xué)功能的探究是對(duì)基因表達(dá)進(jìn)行調(diào)控的重要手段。本研究以番茄(Solanum lycopersicum)Micro-Tom為試驗(yàn)材料,通過常規(guī)PCR的方法克隆得到SlSWEET以基因啟動(dòng)子,進(jìn)一步通過5'端缺失的方法,通過結(jié)合Gateway技術(shù)與pBGWFS7,0表達(dá)載體融合,驅(qū)動(dòng)GUS基因的表達(dá),運(yùn)用GUS組織化學(xué)染色分析和GUS熒光定量分析,明確啟動(dòng)子表達(dá)模式,從而為全面闡述SlSWEET14基因的功能奠定實(shí)驗(yàn)基礎(chǔ),并且也為SWEETs基因家族的研究和番茄優(yōu)質(zhì)品種育種提供依據(jù)。本研究主要結(jié)果如下:1,對(duì)SlSWEET14啟動(dòng)子進(jìn)行生物信息學(xué)分析,表明SlSWEET14基因啟動(dòng)子除含有高等植物通用核心啟動(dòng)子順式作用元件TATA-BOX、CAAT-BOX,還含有與激素響應(yīng)有關(guān)的順式作用元件如:響應(yīng)水楊酸的順式作用元件TCA-element、響應(yīng)赤霉素的順式作用元件TATC-box、響應(yīng)生長(zhǎng)素的順式作用元件AuxRR-core、響應(yīng)乙烯的順式作用元件ERE;與脅迫相關(guān)的順式作用元件如:響應(yīng)熱脅迫的順式作用元件HSE、與干旱誘導(dǎo)有關(guān)的順式作用元件MBS;與胚乳發(fā)育相關(guān)的順式作用元件Skn-1_motif;與光響應(yīng)有關(guān)的順式作用元件GT1-motif、BoxI、Box4、AE-box等。2,利用農(nóng)桿菌介導(dǎo)的果實(shí)注射法分析了啟動(dòng)子的瞬時(shí)表達(dá)活性,運(yùn)用GUS組織化學(xué)染色分析和GUS熒光定量分析結(jié)果表明綠熟期果實(shí)注射后3天,隨著啟動(dòng)子長(zhǎng)度的逐漸變短,其驅(qū)動(dòng)GUS基因的表達(dá)活性逐漸降低。3,通過對(duì)轉(zhuǎn)基因擬南芥組織特異性GUS染色分析,發(fā)現(xiàn)三個(gè)啟動(dòng)子截?cái)嗥尉茯?qū)動(dòng)GUS基因在擬南芥角果果柄處表達(dá),但最短片段驅(qū)動(dòng)GUS基因在擬南芥根和花中也有大量的表達(dá),即啟動(dòng)子最短片段失去了組織特異性,在SlSWEET14啟動(dòng)子上游-1466bp～-767bp處含有決定果實(shí)特異性表達(dá)的順式作用元件。
[Abstract]:The SWEETs gene family is a newly discovered membrane transporter, which can be involved in the transport of sugar, fructose, glucose and other sugars. Previous studies have shown that SISWEET14 is a specific gene expressed in fruit, and it is involved in transshipment of sugars, but its specific function is not clear. In the process of regulating gene expression, promoters play a key regulatory role. Therefore, exploring the biological functions of gene promoters and cis acting elements is an important way to regulate gene expression. In this study, tomato (Solanum lycopersicum Micro-Tom) as test materials, obtain SlSWEET in gene promoter by cloning method of conventional PCR method, further through the 5'terminal deletion, through the combination of Gateway technology and pBGWFS7,0 fusion expression vector and expression of GUS gene driven by GUS, histochemical staining and GUS fluorescence quantitative analysis, a clear start the sub expression pattern, so as to lay the foundation for a comprehensive exposition of the function of SlSWEET14 gene, and also provides the basis for the research and breeding of high quality varieties of tomato SWEETs gene family. The main results of this study are as follows: 1, the promoter of SlSWEET14 bioinformatics analysis showed that SlSWEET14 gene promoter in higher plants contain common core promoter cis element TATA-BOX, CAAT-BOX, also contains the cis acting elements such as hormone response: salicylic acid TCA-element cis element, gibberellin response TATC-box cis element, cis acting elements in response to auxin response AuxRR-core, ethylene ERE cis element; stress-related cis elements such as: heat stress response cis acting elements of HSE, and drought induced cis elements related to MBS; and the development of endosperm related cis element Skn-1_motif; and light responsive cis elements related to GT1-motif, BoxI, Box4, AE-box etc.. 2, using the fruit injection method of Agrobacterium mediated transient expression analysis of promoter activity by GUS histochemical staining and GUS fluorescence quantitative analysis results showed that 3 days after the injection of mature green fruit, with promoter length becomes shorter, the expression of GUS gene driven activity decreased gradually. 3, the transgenic Arabidopsis tissue specific GUS staining analysis found that three promoter fragments were truncated GUS gene expression in Arabidopsis can drive angle fruit handle office, but the short fragment of GUS gene driven are abundantly expressed in roots and flowers of Arabidopsis thaliana, namely the promoter short lost in tissue specificity. The SlSWEET14 promoter -1466bp ~ -767bp which determines the fruit specific expression of the CIS elements.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S641.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 賀飛燕;閆建俊;白云鳳;馮瑞云;施俊鳳;;啟動(dòng)子的類型及應(yīng)用[J];山西農(nóng)業(yè)科學(xué);2017年01期

2 程姣;黃弈;任春梅;;擬南芥CWINV1啟動(dòng)子GUS表達(dá)載體構(gòu)建及轉(zhuǎn)基因植株的鑒定[J];作物研究;2016年03期

3 李強(qiáng)子;張麗;;溫度對(duì)提取DNA質(zhì)量的影響[J];中國生物制品學(xué)雜志;2016年04期

4 雷建峰;李月;徐新霞;阿爾祖古麗·塔什;蒲艷;張巨松;劉曉東;;棉花不同GbU6啟動(dòng)子截短克隆及功能鑒定[J];作物學(xué)報(bào);2016年05期

5 劉暢;姜晶;韓曉雪;韓佳軒;;植物中SWEET基因家族研究進(jìn)展[J];植物生理學(xué)報(bào);2014年09期

6 玄元虎;朱毅勇;胡一兵;;SWEET蛋白家族研究進(jìn)展[J];中國科學(xué):生命科學(xué);2014年07期

7 許蘭珍;彭愛紅;何永睿;姚利曉;雷天剛;劉小豐;姜國金;鄒修平;陳善春;;異源韌皮部特異啟動(dòng)子在轉(zhuǎn)基因枳中的表達(dá)[J];園藝學(xué)報(bào);2014年01期

8 周丹丹;俞嘉寧;;植物細(xì)胞中瞬時(shí)表達(dá)系統(tǒng)的建立及研究進(jìn)展[J];中國農(nóng)學(xué)通報(bào);2013年24期

9 趙文婷;魏建和;劉曉東;高志暉;;植物瞬時(shí)表達(dá)技術(shù)的主要方法與應(yīng)用進(jìn)展[J];生物技術(shù)通訊;2013年02期

10 魏晶;毛偉華;林擁軍;陳浩;;1個(gè)新水稻組成型啟動(dòng)子的克隆與功能鑒定[J];華中農(nóng)業(yè)大學(xué)學(xué)報(bào);2012年02期

相關(guān)博士學(xué)位論文 前2條

1 王鳳婷;玉米逆境脅迫誘導(dǎo)型啟動(dòng)子PZmCKS2、PZmCBF3及轉(zhuǎn)錄因子ZmZBED的功能解析[D];吉林大學(xué);2015年

2 孔令廣;xa13基因在水稻—白葉枯病菌互作過程中的功能研究[D];山東農(nóng)業(yè)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 吳憲;擬南芥干旱誘導(dǎo)型啟動(dòng)子的克隆及功能分析[D];吉林大學(xué);2013年

2 何佳;調(diào)控?cái)M南芥雄性不育基因MA的定位及擬南芥MtN3/saliva基因家族分析[D];上海師范大學(xué);2010年

，

本文編號(hào)：1339923