發(fā)布時(shí)間：2017-12-28 00:31

  本文關(guān)鍵詞：岷江百合GPAT基因啟動(dòng)子的克隆與功能元件初步分析　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： GPAT基因啟動(dòng)子 啟動(dòng)子克隆 接頭PCR 順式作用元件 煙草轉(zhuǎn)化 缺失重組表達(dá)載體

【摘要】：現(xiàn)今社會(huì),研究環(huán)境脅迫對植物生長發(fā)育的影響已經(jīng)成為生物學(xué)術(shù)界的熱門。運(yùn)用基因技術(shù)等手段提高植物的抗逆性已成為方法之一。啟動(dòng)子是調(diào)控基因表達(dá)的關(guān)鍵。所以克隆和構(gòu)建環(huán)境誘導(dǎo)型啟動(dòng)子是培育植物抗逆品種的根本途徑�；诮┠甑难芯拷Y(jié)果,諸如CaMV35S、玉米Ubiquitinpromoter等啟動(dòng)子已經(jīng)被廣泛應(yīng)用。但是這些啟動(dòng)子的特性決定著它們在植物的整個(gè)生長發(fā)育時(shí)期都會(huì)高水平表達(dá),不僅會(huì)造成資源浪費(fèi),嚴(yán)重時(shí)會(huì)使植物致死,無法達(dá)到高效,可調(diào)控(定時(shí)、定點(diǎn)、定量)表達(dá)。而誘導(dǎo)型啟動(dòng)子可以誘導(dǎo)植物在特定的時(shí)期、特定條件下表達(dá),避免了不必要的資源浪費(fèi),也降低了給植物造成的有害影響。因此誘導(dǎo)型啟動(dòng)子的探究和應(yīng)用成為研究者們追逐的新潮。本研究基于前人和課題組研究的結(jié)果,選取岷江百合作為試驗(yàn)材料,利用有效的基因克隆技術(shù),克隆岷江百合GPAT基因的啟動(dòng)子,并對該啟動(dòng)子做了初步的表達(dá)分析,主要結(jié)果如下:1.基于本課題組研究得到的岷江百合GPAT基因序列,在該基因編碼區(qū)設(shè)計(jì)基因特異性引物,以岷江百合基因組DNA為模板,運(yùn)用融合嵌套PCR和接頭PCR技術(shù)克隆岷江百合GPAT基因啟動(dòng)子,共步移得到1007bp的啟動(dòng)子序列,命名為GPATp。2.對步移得到的1007匕p啟動(dòng)子序列進(jìn)行序列分析,利用NewPLACE、PlantARE等分析預(yù)測網(wǎng)站,結(jié)果發(fā)現(xiàn)在序列中包含許多的TATA-box、CAAT-box等核心順式元件,另外還有許多與環(huán)境脅迫相關(guān)的順式作用元件和組織特異性元件。3.利用缺失克隆,并以GUS為報(bào)告基因?qū)?dòng)子進(jìn)行表達(dá)分析,構(gòu)建了 5個(gè)缺失啟動(dòng)子片段:GPATp1(954bp)、GPATp2(644bp)、GPATp3(386bp)、GPATp4(231 bp)、GPATp5(169 bp),分別將這些啟動(dòng)子片段替換植物表達(dá)載體pCAMBIA 3301中的35S啟動(dòng)子,構(gòu)建了 5個(gè)植物重組表達(dá)載體,分別命名為GPATp1～5.4.煙草轉(zhuǎn)化:利用農(nóng)桿菌介導(dǎo)的葉盤轉(zhuǎn)化法轉(zhuǎn)化煙草葉盤,以GUS基因?yàn)閳?bào)告基因進(jìn)行瞬時(shí)表達(dá)分析,結(jié)果顯示:該GPATp5(169 bp)組葉盤沒有被染成藍(lán)色,說明該啟動(dòng)子片段不能調(diào)控GUS基因表達(dá),啟動(dòng)子沒有活性;GPATp4(231 bp)組在其他四組之中的表達(dá)最弱,結(jié)合啟動(dòng)子預(yù)測分析結(jié)果推測該片段是最小的具有啟動(dòng)子活性的片段;GPATp3(386bp)組比GPATp4(231 bp)組相對染色較深,但弱于GPATp2(644 bp)組和 GPATp1(954 bp)組;GPATp2(644 bp)組深于 GPATp3(386 bp)組;GPATp1(954 bp)組顏色最深,說明該片段活性最強(qiáng)。在進(jìn)行缺失重組載體轉(zhuǎn)化煙草的同時(shí),每組轉(zhuǎn)化分別進(jìn)行常溫(25 ℃)和低溫處理(4℃),結(jié)果發(fā)現(xiàn):GPATp1(954 bp)組和 GPATp2(644 bp)組在 4 ℃處理一段時(shí)間之后,相對同組常溫葉盤顏色略微加深,但不明顯,啟動(dòng)子預(yù)測分析顯示在該啟動(dòng)子片段中含有冷相關(guān)順式元件,說明該啟動(dòng)子在一定程度上受冷誘導(dǎo),但具體誘導(dǎo)情況還需進(jìn)一步研究。
[Abstract]:Nowadays, the study of the influence of environmental stress on the growth and development of plants has become a hot topic in the academic community. It has become one of the methods to improve the resistance of plants by means of gene technology. Promoter is the key to regulate gene expression. Therefore, cloning and construction of environmental inducible promoters is the fundamental way to cultivate plant resistant varieties. Based on recent research results, such as CaMV35S, maize Ubiquitinpromoter and other promoters have been widely used. However, the characteristics of these promoters decide that they will be highly expressed in the whole growth stage of plants, which will not only cause waste of resources, but also make plants lethal, and can not achieve efficient, controllable (timing, fixed-point, quantitative) expression. Inducible promoters can induce plants to express at specific times and under specific conditions, avoiding unnecessary waste of resources and reducing harmful effects on plants. Therefore, the exploration and application of the inducible promoter has become a new trend for researchers. This study is based on previous research results and the research group, select the l.regale as experimental materials, the effective use of gene cloning technology, cloning of l.regale promoter of GPAT gene, and the promoter made a preliminary analysis of the expression, the main results are as follows: 1. based on the GPAT gene sequence of l.regale group get this subject. In the design of the gene encoding gene specific primers to l.regale genomic DNA as template, using nested PCR and PCR fusion joint l.regale cloning GPAT gene promoter, were walking by the 1007bp promoter sequence, named GPATp. The 2. step of the 1007 point P promoter sequence and the sequence analysis, prediction of site analysis by NewPLACE and PlantARE, were found in the sequence contains many TATA-box, CAAT-box and other core cis elements, as well as many other related environmental stress cis acting elements and tissue specific element. 3. with the deletion clones, and with GUS as reporter gene expression analysis of promoter constructs 5 deletion promoter fragments: GPATp1 (954bp), GPATp2 (644bp), GPATp3 (386bp), GPATp4 (231 BP), GPATp5 (169 BP), respectively, the promoter fragment replacement plant expression vector pCAMBIA 3301 in the 35S promoter, constructed 5 plant expression vector, respectively named GPATp1 ~ 5.4.: Tobacco by Agrobacterium mediated leaf disc transformation of tobacco leaf disc, using GUS gene as a reporter gene expression analysis, the results showed that: the GPATp5 group (169 BP) leaf disc not be dyed blue, that expression of the promoter fragment of GUS gene promoter is not regulated, no activity; GPATp4 (231 BP) group in the other four groups in the lowest expression and binding to the promoter prediction analysis results suggest that this fragment is the small promoter activity of GP fragment; The ATp3 (386bp) group is relatively darker than that of GPATp4 (231 BP) group, but weaker than that of GPATp2 (644 BP) group and GPATp1 (954 BP) group. GPATp2 (644 BP) group is deeper than that of GPATp3 (386 GPATp2) group, and the color group is the most deep, indicating that the fragment has the strongest activity. In the absence of the recombinant vector into tobacco at the same time, each transformation respectively at room temperature (25 DEG C) and low temperature (4 DEG C), the results showed that: GPATp1 (954 BP) group and GPATp2 (644 BP) group after 4 treatment for a period of time, relative to the same group of normal leaf disc color slightly deep, but no obvious promoter prediction analysis showed that the promoter fragment contains cold related cis elements, indicating that the promoter induced by cold to a certain extent, but the specific induction needs further study.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S682.29

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