本文選題：轉(zhuǎn)基因煙草 + 玉米ZmPIS啟動(dòng)子�。� 參考：《山東大學(xué)》2014年碩士論文

【摘要】：土壤干旱和鹽漬化等嚴(yán)重限制了植物的生長(zhǎng)發(fā)育,已成為影響作物品質(zhì)和產(chǎn)量的重要因素。作物應(yīng)對(duì)干旱和土壤鹽漬化不良環(huán)境,促進(jìn)產(chǎn)量的生物學(xué)過(guò)程受到包含多個(gè)關(guān)鍵基因在內(nèi)的協(xié)同調(diào)控過(guò)程。發(fā)掘并克隆耐逆關(guān)鍵功能基因及調(diào)節(jié)元件,采用轉(zhuǎn)基因技術(shù)培育抗旱、耐鹽作物新品種,是解決上述問(wèn)題的有效途徑。目前,利用植物基因工程手段改良作物相關(guān)性狀時(shí),轉(zhuǎn)化的目標(biāo)基因在植物宿主細(xì)胞中的高效表達(dá)和組織或時(shí)空特異性等受到越來(lái)越多的關(guān)注�；虻谋磉_(dá)調(diào)控是在DNA水平、轉(zhuǎn)錄水平、翻譯及翻譯后修飾水平等多層次協(xié)同調(diào)控的過(guò)程,其中轉(zhuǎn)錄水平的調(diào)控主要是通過(guò)啟動(dòng)子上的順式作用元件與反式作用因子的相互作用來(lái)實(shí)現(xiàn)功能基因在特定的時(shí)間、組織中表達(dá)。分離克隆可用于作物抗逆遺傳改良的啟動(dòng)子并獲得自主知識(shí)產(chǎn)權(quán),了解各啟動(dòng)子中的核心功能區(qū)段及調(diào)控元件的功能基礎(chǔ)具有重要的理論意義和應(yīng)用價(jià)值。在實(shí)驗(yàn)室前期工作中,翟淑梅等發(fā)現(xiàn)玉米ZmPIS基因具有明顯的鹽和干旱等脅迫誘導(dǎo)特性。官贊贊等克隆了ZmPIS基因的啟動(dòng)子序列并初步確定了該啟動(dòng)子啟動(dòng)目的基因表達(dá)的能力。本工作在上述工作基礎(chǔ)上對(duì)ZmPIS基因的啟動(dòng)子的功能及脅迫響應(yīng)特性進(jìn)行了系統(tǒng)地分析。ZmPIS基因啟動(dòng)子的生物信息學(xué)分析表明該啟動(dòng)子含有多個(gè)脅迫響應(yīng)元件。我們構(gòu)建了該啟動(dòng)子的5’端系列缺失突變植物表達(dá)載體PZ1-PZ8,啟動(dòng)Gus報(bào)告基因的表達(dá),轉(zhuǎn)化煙草。對(duì)含有啟動(dòng)子不同長(zhǎng)度片段的轉(zhuǎn)基因煙草葉片進(jìn)行了GUS熒光活性測(cè)定,發(fā)現(xiàn)PZ7在PZ1-PZ8轉(zhuǎn)基因煙草葉片材料中表現(xiàn)出了最高的啟動(dòng)目的基因表達(dá)的能力。以PZ1、PZ7和35S啟動(dòng)子轉(zhuǎn)基因煙草2周齡植株及花、果、種子進(jìn)行GUS組織化學(xué)染色,發(fā)現(xiàn)PZ7相對(duì)于PZ1全長(zhǎng)啟動(dòng)子在上述不同組織器官中均具有更高的GUS表達(dá)強(qiáng)度。為進(jìn)一步研究ZmPIS啟動(dòng)子脅迫響應(yīng)特性,我們對(duì)PZ1-PZ8轉(zhuǎn)基因煙草進(jìn)行了NaCl和18% PEG等脅迫處理分析。轉(zhuǎn)基因煙草離體葉片在200mMNaCl、18% PEG處理下的GUS組織化學(xué)染色結(jié)果表明,鹽和滲透脅迫下PZ1-PZ7有相似的誘導(dǎo)表達(dá)模式,即處理1 h、3 h、6 h時(shí)與未處理的對(duì)照相比無(wú)顯著差異,12 h時(shí)表現(xiàn)出一定誘導(dǎo)趨勢(shì),處理至24 h、48 h和72 h時(shí)GUS染色強(qiáng)度顯著上調(diào),且PZ7在脅迫誘導(dǎo)前后均具有最高GUS表達(dá)強(qiáng)度。相對(duì)而言PZ8和35S啟動(dòng)子在上述兩種脅迫環(huán)境下均未表現(xiàn)出明顯的誘導(dǎo)表達(dá)變化。為進(jìn)一步驗(yàn)證上述離體實(shí)驗(yàn)的結(jié)果,我們以PZ1、PZ2、PZ6、PZ7、PZ8、35S轉(zhuǎn)基因煙草活體植株為材料開(kāi)展了相應(yīng)的NaCl和PEG脅迫處理實(shí)驗(yàn)。GUS組織化學(xué)染色及熒光活性測(cè)定結(jié)果表明,處理24 h時(shí)PZ1、PZ2、PZ6和PZ7轉(zhuǎn)基因煙草中的GUS酶活性達(dá)到最高(均達(dá)到處理前的2倍以上),且在處理48 h和72 h時(shí)一致保持較高的誘導(dǎo)水平,也就是說(shuō)PZ1-PZ7均具有明顯的高鹽和滲透脅迫誘導(dǎo)活性,而PZ8的高鹽和滲透脅迫誘導(dǎo)活性卻明顯喪失。值得注意的是PZ7在脅迫前后與其它突變體材料相比均體現(xiàn)出了最高啟動(dòng)活性,可達(dá)到PZ1(全長(zhǎng)啟動(dòng)子)的20倍以上,PZ6的3倍以上。正常條件下PZ7的啟動(dòng)表達(dá)活性為PZ8的1.1倍左右和35S啟動(dòng)子的20%左右；而鹽和滲透脅迫后可達(dá)到PZ8的2倍以上和35S啟動(dòng)子50%左右。上述結(jié)果意味著PZ7-PZ8缺失的110 bp片段(-466到-357 bp)中可能含有高鹽和滲透脅迫響應(yīng)元件,且PZ7(-466 bp片段)為ZmPIS啟動(dòng)子具有鹽和滲透脅迫響應(yīng)特性的高效啟動(dòng)基因表達(dá)的關(guān)鍵序列區(qū)段,體現(xiàn)出了一定的應(yīng)用潛質(zhì)。本工作通過(guò)對(duì)玉米ZmPIS啟動(dòng)子的功能及脅迫響應(yīng)特性分析,發(fā)現(xiàn)了PZ7(-466 bp)片段為該啟動(dòng)子具有鹽和滲透脅迫響應(yīng)活性的高效啟動(dòng)基因表達(dá)的核心功能區(qū)段,而-466 bp到-357 bp之間的110 bp序列可能存在鹽和滲透脅迫響應(yīng)順式作用元件。這些實(shí)驗(yàn)結(jié)果加深了我們對(duì)玉米ZmPIS基因啟動(dòng)子上核心功能區(qū)段及脅迫響應(yīng)特性的了解,為作物抗逆基因工程育種提供了一定的啟動(dòng)子資源和參考資料,有望為我國(guó)作物抗逆遺傳改良貢獻(xiàn)自己的綿薄之力。
[Abstract]:Soil drought and salinization have seriously restricted the growth and development of plants. It has become an important factor affecting the quality and yield of crops. The biological process of crop response to drought and soil salinization and the biological process of promoting yield are coordinated by several key genes. The use of transgenic technology to cultivate drought resistant and salt tolerant crop varieties is an effective way to solve the above problems. At present, more and more attention has been paid to the efficient expression and tissue or time space specificity of the target gene in plant host cells by using plant genetic engineering methods to improve crop correlation. Regulation is a multi level coordination process, such as DNA level, transcriptional level, translation and post-translational modification. The regulation of transcriptional level is mainly through the interaction of the cis acting element and the trans acting factor on the promoter to realize the functional genes in a specific time and tissue. The isolated clones can be used for crop resistance. In the early work of the laboratory, Zhai Shumei found that the maize ZmPIS gene had obvious salt and drought stress induction characteristics. The promoter sequence of the ZmPIS gene was augmentation and the ability of the promoter to start the gene expression was preliminarily determined. On the basis of the above work, a systematic analysis of the biological information of the promoter of the.ZmPIS gene was carried out on the basis of the function of the promoter of the ZmPIS gene and the response characteristics of the promoter, and it was shown that the promoter contains multiple stress responses. Components. We constructed a 5 'terminal series deletion mutant plant expression vector PZ1-PZ8, started the expression of Gus reporter gene and transformed tobacco. The GUS fluorescence activity of transgenic tobacco leaves with different lengths of promoter was measured. It was found that PZ7 showed the highest start in the PZ1-PZ8 transgenic tobacco leaf material. The ability of the target gene expression. The 2 weeks old plants of PZ1, PZ7 and 35S promoters, flowers, fruits and seeds were stained with GUS histochemical staining. It was found that PZ7 had a higher GUS expression intensity in the different tissues and organs of the PZ1. To further study the response characteristics of the ZmPIS promoter stress, we had the PZ1-PZ8 transgene. The NaCl and 18% PEG stress treatments of tobacco were analyzed. The GUS histochemical staining results of the leaves of transgenic tobacco under the treatment of 200mMNaCl and 18% PEG showed that the PZ1-PZ7 had similar induced expression patterns under salt and osmotic stress, that is, the treatment of 1 h, 3 h, 6 h was not significantly different from that of the untreated, while 12 h showed a certain inducement. The guiding trend, when treated to 24 h, 48 h and 72 h, the GUS staining intensity was significantly up-regulated, and PZ7 had the highest GUS expression intensity before and after stress induction. Relatively speaking, PZ8 and 35S promoter did not show obvious inducible expression changes under these two stress environments. The Z8,35S transgenic tobacco living plants carried out the corresponding NaCl and PEG stress treatment experiments. The results of histochemical staining and fluorescence activity of.GUS showed that the activity of GUS enzyme in PZ1, PZ2, PZ6 and PZ7 transgenic tobacco reached the highest when treating 24 h (all reached more than 2 times before the treatment), and remained high in the treatment of 48 h and 72 h. The induction level, that is, PZ1-PZ7 has obvious high salt and osmotic stress induction activity, while the high salt and osmotic stress induced activity of PZ8 is lost obviously. It is worth noting that PZ7 shows the highest activation activity compared with other mutant materials before and after stress, and can reach more than 20 times of PZ1 (full promoter), and 3 times of PZ6. Above. Under normal conditions, the activation of PZ7 is about 1.1 times of PZ8 and about 20% of 35S promoter, while salt and osmotic stress can reach more than 2 times of PZ8 and 50% of 35S promoter. The above results mean that the 110 BP fragments of PZ7-PZ8 deletion (-466 to -357 BP) may contain high salt and osmotic stress response elements, and PZ7 (-466) Fragment) is the key sequence section of high efficient promoter gene expression of ZmPIS promoter with salt and osmotic stress response characteristics, reflecting certain application potential. This work has found that PZ7 (-466 BP) fragment has salt and osmotic response activity through the analysis of the function and stress response characteristics of maize ZmPIS promoter. The 110 BP sequences between -466 BP and -357 BP may have salt and osmotic response cis elements. These results deepen our solution to the core functional areas and stress response characteristics of the maize ZmPIS promoter, and provide the crop resistant genetic engineering breeding. Certain promoter resources and reference materials are expected to contribute to the improvement of crop genetic resistance in China.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：Q943.2

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