本文選題：營(yíng)養(yǎng)器官 + 啟動(dòng)子�。� 參考：《安徽農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：基因工程中能否使外源基因以一定的表達(dá)量在特定的組織部位表達(dá)是一個(gè)重要問(wèn)題,而啟動(dòng)子作為能夠調(diào)控外源基因在特定組織器官表達(dá)的分子元件之一,具有重要的研究意義。目前人們普遍使用的是組成型啟動(dòng)子,這類啟動(dòng)子能驅(qū)動(dòng)外源基因在植物中各部位高效的表達(dá),但同時(shí)也造成了營(yíng)養(yǎng)的不必要浪費(fèi)以及植物細(xì)胞的過(guò)度負(fù)荷。特異性啟動(dòng)子可以通過(guò)調(diào)控目的基因使其在特定的組織部位表達(dá)。因此,對(duì)玉米中特異性啟動(dòng)子的研究不僅對(duì)改善玉米的品質(zhì)具有重要意義,而且為基因工程中調(diào)控元件的研究提供了參考。本課題在生物信息學(xué)的分析結(jié)果的基礎(chǔ)上,構(gòu)建了玉米CCR基因啟動(dòng)子以及其篩檢片段的表達(dá)載體,通過(guò)農(nóng)桿菌介導(dǎo)法獲得轉(zhuǎn)基因的水稻植株,并對(duì)其表達(dá)特性進(jìn)行了分析,獲得如下主要研究結(jié)果:1.通過(guò)啟動(dòng)子序列分析的相關(guān)網(wǎng)站結(jié)果顯示,該啟動(dòng)子除了基本順式作用元件TATA-box和CAAT-box外,還含有四種誘導(dǎo)型元件:ARE、Box-W1、CGTCA-motif和MBS;四種光應(yīng)答元件Box 4、Box I、G-box和Sp1;三種器官表達(dá)元件ROOTMOTIFTAPOX1、POLLEN1LELAT52和Skn-1。2.從玉米基因組中分離出一段2145bp的肉桂酰輔酶A還原酶(cinnamoyl-CoA reductase)基因啟動(dòng)子,將其命名為pCCR。通過(guò)5’端缺失法對(duì)CCR基因啟動(dòng)子的全長(zhǎng)進(jìn)行片段篩檢,得到三個(gè)不同長(zhǎng)度的篩檢片段我們分別將其命名為pCCR-1、pCCR-2以及pCCR-3,其片段大小分別為1650 bp、1126 bp以及548 bp。通過(guò)雙酶切的方法將這四個(gè)啟動(dòng)子片段與含有GUS報(bào)告基因的雙元表達(dá)載體pCAMBIA1301連接,構(gòu)建四個(gè)新型表達(dá)載體。3.將構(gòu)建的四個(gè)新型表達(dá)載體運(yùn)用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)入水稻中花11中,共獲得42株轉(zhuǎn)基因植株苗,其中CCR有11株,CCR-1有13株,CCR-2有8株,CCR-3有10株。通過(guò)PCR及潮霉素檢測(cè)后,共有27株為陽(yáng)性植株。4.采用GUS組織化學(xué)染色方法,結(jié)果顯示CCR基因啟動(dòng)子在根、莖、葉、穎殼中表達(dá),花和種子不表達(dá),推測(cè)其為營(yíng)養(yǎng)器官特異性啟動(dòng)子;篩減片段pCCR-1在葉、穎殼、花以及胚中都有表達(dá),其余部位不表達(dá);pCCR-2只在葉中表達(dá),其余組織部位均不表達(dá),即pCCR-2為葉特異性啟動(dòng)子;pCCR-3在莖與穎殼部位表達(dá),其余部位不表達(dá);
[Abstract]:Whether the exogenous gene can be expressed in a certain amount in specific tissues is an important issue in genetic engineering, and promoter is one of the molecular elements that can regulate the expression of exogenous gene in specific tissues and organs.It has important research significance.At present, constitutive promoters are widely used, which can drive the efficient expression of exogenous genes in various parts of plants, but also lead to unnecessary waste of nutrition and excessive loading of plant cells.Specific promoters can be expressed in specific tissues by regulating the target gene.Therefore, the study of specific promoters in maize is of great significance not only to improve the quality of maize, but also to provide a reference for the study of regulatory elements in genetic engineering.Based on the results of bioinformatics analysis, the expression vector of maize CCR gene promoter and its screening fragment was constructed. The transgenic rice plants were obtained by Agrobacterium tumefaciens mediated method, and their expression characteristics were analyzed.The following main results are obtained: 1: 1.In addition to the basic cis-acting elements TATA-box and CAAT-box, the promoter also contains four inductive elements, namely: AREX Box-W _ (1) CGTCA-motif and MBSs; four optical response elements, Box _ 4, Box Ion G-box and Sp1; and three organ expression elements, ROOTMOTIFTAPOX1 POLLEN1LEAT52 and Skn-1.2.A 2145bp cinnamoyl-CoA reductase promoter was isolated from maize genome and named pCCR.The full-length of CCR promoter was screened by 5'end deletion method, and three screening fragments of different length were obtained. We named them pCCR-1pCCR-2 and pCCR-3, respectively, with the size of 1650 BP and 548 BP, respectively.The four promoter fragments were ligated with the dual expression vector pCAMBIA1301 containing GUS reporter gene, and four novel expression vectors. 3 were constructed by double enzyme digestion.The four new expression vectors were transferred into rice Zhonghua 11 by Agrobacterium tumefaciens, and 42 transgenic plants were obtained, of which 11 were CCR-1 and 13 were CCR-2, 8 were CCR-3 and 10 were CCR.After PCR and hygromycin detection, there were 27 positive plants.The results of GUS histochemical staining showed that the promoter of CCR gene was expressed in root, stem, leaf and glume, but was not expressed in flower and seed.There was no expression of pCCR-2 in the other parts of flower and embryo, but no expression of pCCR-2 in other tissues. That is to say, pCCR-2 was expressed in stem and glume, but not in other parts.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S513;Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陶興林;;GUS基因在農(nóng)桿菌和甜瓜中的表達(dá)差異研究[J];北方園藝;2008年04期

2 于翠梅;馬蓮菊;張寶石;;特異性啟動(dòng)子在植物基因工程中的應(yīng)用[J];生物工程學(xué)報(bào);2006年06期

3 朱青;宋波濤;柳俊;謝從華;;擬南芥低溫誘導(dǎo)cor15a基因的啟動(dòng)子克隆及在轉(zhuǎn)基因馬鈴薯中的表達(dá)[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2004年05期

4 曾海濤,王義琴,陳英,潘惠新,黃敏仁;花卉花色基因工程的研究現(xiàn)狀及存在問(wèn)題[J];中國(guó)生物工程雜志;2004年06期

5 王穎,麥維軍,梁承鄴,張明永;高等植物啟動(dòng)子的研究進(jìn)展[J];西北植物學(xué)報(bào);2003年11期

6 孫曉紅,陳明杰,潘迎捷;啟動(dòng)子克隆概述[J];食用菌學(xué)報(bào);2002年03期

7 李竹紅,劉德培,梁植權(quán);靶向整合研究進(jìn)展[J];生物化學(xué)與生物物理學(xué)報(bào);1999年01期

8 張曉偉,王福山,童坦君;綠色熒光蛋白cDNA在腺病毒重組載體轉(zhuǎn)染中的應(yīng)用[J];生物化學(xué)與生物物理進(jìn)展;1998年03期

9 郭元林,向平;轉(zhuǎn)基因技術(shù)在作物育種上的應(yīng)用[J];西南農(nóng)業(yè)學(xué)報(bào);1997年04期

10 鞏振輝，何玉科;擬南芥基因轉(zhuǎn)移新方法一真空滲入法的研究[J];西北植物學(xué)報(bào);1996年03期

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