本文選題：營養(yǎng)器官 + 啟動子��； 參考：《安徽農(nóng)業(yè)大學》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量在特定的組織部位表達是一個重要問題,而啟動子作為能夠調(diào)控外源基因在特定組織器官表達的分子元件之一,具有重要的研究意義。目前人們普遍使用的是組成型啟動子,這類啟動子能驅(qū)動外源基因在植物中各部位高效的表達,但同時也造成了營養(yǎng)的不必要浪費以及植物細胞的過度負荷。特異性啟動子可以通過調(diào)控目的基因使其在特定的組織部位表達。因此,對玉米中特異性啟動子的研究不僅對改善玉米的品質(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的分析結(jié)果的基礎上,構(gòu)建了玉米CCR基因啟動子以及其篩檢片段的表達載體,通過農(nóng)桿菌介導法獲得轉(zhuǎn)基因的水稻植株,并對其表達特性進行了分析,獲得如下主要研究結(jié)果:1.通過啟動子序列分析的相關(guān)網(wǎng)站結(jié)果顯示,該啟動子除了基本順式作用元件TATA-box和CAAT-box外,還含有四種誘導型元件:ARE、Box-W1、CGTCA-motif和MBS;四種光應答元件Box 4、Box I、G-box和Sp1;三種器官表達元件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)基因啟動子,將其命名為pCCR。通過5’端缺失法對CCR基因啟動子的全長進行片段篩檢,得到三個不同長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。通過雙酶切的方法將這四個啟動子片段與含有GUS報告基因的雙元表達載體pCAMBIA1301連接,構(gòu)建四個新型表達載體。3.將構(gòu)建的四個新型表達載體運用農(nóng)桿菌介導法轉(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PCR及潮霉素檢測后,共有27株為陽性植株。4.采用GUS組織化學染色方法,結(jié)果顯示CCR基因啟動子在根、莖、葉、穎殼中表達,花和種子不表達,推測其為營養(yǎng)器官特異性啟動子;篩減片段pCCR-1在葉、穎殼、花以及胚中都有表達,其余部位不表達;pCCR-2只在葉中表達,其余組織部位均不表達,即pCCR-2為葉特異性啟動子;pCCR-3在莖與穎殼部位表達,其余部位不表達;
[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.
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S513;Q943.2

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