本文選題：鹽生草 + HgNHX1基因��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鹽脅迫是全球范圍內(nèi)重要的非生物脅迫之一,它會(huì)使植物所處的土壤環(huán)境Na~+濃度增高,從而導(dǎo)致植物離子穩(wěn)態(tài)被破壞并發(fā)生滲透脅迫。而液泡膜Na~+/H~+逆向轉(zhuǎn)運(yùn)蛋白(NHX)對(duì)植物體的離子平衡、耐鹽性以及植株的整個(gè)發(fā)育過(guò)程都起著很重要的作用。鹽生草(Halogeton glomeratus)為藜科(Chenopodiaceae)鹽生草屬一年生雙子葉植物,廣泛分布于我國(guó)西北旱區(qū)及半干旱區(qū),其莖、葉均高度肉質(zhì)化,可以在鹽堿化環(huán)境中正常生長(zhǎng),是挖掘植物耐鹽基因及研究耐鹽調(diào)控機(jī)理的良好材料,對(duì)改良作物抗逆性及培育耐鹽堿作物具有重要的應(yīng)用價(jià)值。其最主要的耐鹽機(jī)制是將鹽毒性離子區(qū)隔化在細(xì)胞液泡中。因此,可在分子水平研究其耐鹽機(jī)理。基因工程中以35S啟動(dòng)子的使用最為廣泛,它能夠滿足在植物組織器官中非特異性高效表達(dá),但是這種表達(dá)方式會(huì)打破植物原有的代謝平衡,從而影響植物的正常生長(zhǎng)。為了減少這種不利影響,克隆新的具有組織特異性的啟動(dòng)子在基因工程的研究與發(fā)展中意義深遠(yuǎn)。本實(shí)驗(yàn)以鹽生草為材料克隆了HgNHX1基因上游的啟動(dòng)子(pHgNHX1),并對(duì)pHgNHX1的功能進(jìn)行了探索;同時(shí)對(duì)HgNHX1基因轉(zhuǎn)化擬南芥后進(jìn)行耐鹽抗旱功能研究。主要結(jié)果如下:1.根據(jù)已獲得的pHgNHX1序列設(shè)計(jì)上下游引物,克隆HgNHX1基因上游的啟動(dòng)子,并利用軟件PlantCARE和PLACE等對(duì)pHgNHX1進(jìn)行功能元件的分析,發(fā)現(xiàn)pHgNHX1中除具有核心啟動(dòng)子元件外還包含響應(yīng)逆境脅迫誘導(dǎo)和植物激素誘導(dǎo)的功能元件,說(shuō)明該啟動(dòng)子具有典型啟動(dòng)子的一般特征。2.構(gòu)建缺失HgNHX1基因上游不同長(zhǎng)度的啟動(dòng)子表達(dá)載體pBI-pHgNHX1并轉(zhuǎn)化煙草和擬南芥,對(duì)陽(yáng)性克隆植株進(jìn)行GUS組織化學(xué)染色,結(jié)果表明:4個(gè)不同長(zhǎng)度片段的啟動(dòng)子都可驅(qū)使GUS基因在煙草葉片和擬南芥株系中表達(dá),說(shuō)明該啟動(dòng)子具有啟動(dòng)子活性,并且在HgNHX1基因上游611 bp得片段就包含啟動(dòng)子的核心表達(dá)元件,但?1染色結(jié)果較其他三個(gè)片段稍淺。3.對(duì)已成功轉(zhuǎn)化了HgNHX1基因上游1523 bp啟動(dòng)子的煙草和擬南芥不同組織進(jìn)行GUS染色,結(jié)果顯示在煙草的葉和莖、擬南芥不同生長(zhǎng)時(shí)期、不同組織中均可顯色,說(shuō)明該啟動(dòng)子具有組成型啟動(dòng)子的特性。4.將本實(shí)驗(yàn)室構(gòu)建的表達(dá)載體pCAMBIA1301-HgNHX1利用浸花法轉(zhuǎn)化擬南芥,種植陽(yáng)性T2代植株并進(jìn)行耐鹽抗旱功能研究,結(jié)果顯示在干旱及鹽脅迫下該基因表達(dá)量雖然增加了,但是通過(guò)表型變化的觀察及相關(guān)生理指標(biāo)的測(cè)定發(fā)現(xiàn)轉(zhuǎn)基因擬南芥的抗旱性和耐鹽性并沒(méi)有提高。
[Abstract]:Salt stress is one of the most important abiotic stresses in the world. It can increase the concentration of Na ~ in the soil environment of plants, which leads to the destruction of plant ion homeostasis and osmotic stress. The vacuolar membrane Na- / H- antiporter (NHX) plays an important role in ion balance, salt tolerance and plant development. Halophyte glomeratus, an annual dicotyledonous plant of Chenopodiaceae, is widely distributed in arid and semi-arid regions of northwest China. Its stems and leaves are highly fleshy and can grow normally in saline-alkali environment. It is a good material to excavate plant salt tolerance genes and study the mechanism of salt tolerance. It has important application value in improving crop stress resistance and cultivating saline-alkali tolerant crops. The main mechanism of salt tolerance is to separate salt toxic ions into vacuoles. Therefore, the mechanism of salt tolerance can be studied at molecular level. In genetic engineering, 35s promoter is the most widely used, which can satisfy the non-specific and high-efficiency expression in plant tissues and organs, but this expression will upset the original metabolic balance of plants, thus affecting the normal growth of plants. In order to reduce this adverse effect, the cloning of new tissue specific promoters is of great significance in the research and development of genetic engineering. The promoter of HgNHX1 gene upstream (pHgNHX1) was cloned and the function of pHgNHX1 was explored, and the salt-tolerant and drought-resistant function of HgNHX1 gene transformed into Arabidopsis thaliana was studied. The main results are as follows: 1. The upstream promoter of the HgNHX1 gene was cloned according to the sequence of pH gNHX1, and the functional components of the HgNHX1 gene were analyzed by using the software Plantcare and place. It was found that pHgNHX1 not only has the core promoter element, but also contains the functional elements in response to stress and phytohormone induction, indicating that the promoter has the general characteristics of typical promoter. 2. The promoter expression vector pBI-pHgNHX1 was constructed and transformed into tobacco and Arabidopsis thaliana. The positive clones were stained with Gus histochemistry. The results showed that Gus gene could be expressed in tobacco leaves and Arabidopsis thaliana lines by four different length promoters, which indicated that the promoter had promoter activity. The 611bp fragment upstream of the HgNHX1 gene contained the core expression elements of the promoter, but the staining results of HgNHX1 were slightly lighter than those of the other three fragments. Different tissues of tobacco and Arabidopsis thaliana which have been successfully transformed into 1523 BP promoter upstream of HgNHX1 gene were stained with Gus. The results showed that different tissues could be stained in leaves and stems of tobacco, different growth stages of Arabidopsis thaliana, and different tissues of Arabidopsis thaliana. It shows that the promoter has the characteristic of component promoter. 4. The expression vector pCAMBIA1301-HgNHX1 was used to transform Arabidopsis thaliana into Arabidopsis thaliana by blooming method. The results showed that the expression of pCAMBIA1301-HgNHX1 was increased under drought and salt stress. However, the observation of phenotypic changes and the determination of related physiological indexes showed that the drought resistance and salt tolerance of transgenic Arabidopsis thaliana were not improved.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2

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