紫花苜蓿玉米黃質(zhì)環(huán)氧化酶基因（MsZEP）的克隆及功能研究
發(fā)布時間：2018-08-07 08:03
【摘要】：紫花苜蓿(Medicago sativa L.)是一種能夠通過根瘤生物固氮的高品質(zhì)豆科牧草,具有栽培范圍廣、適應(yīng)性強(qiáng)、品質(zhì)好、產(chǎn)量高等優(yōu)點,被譽(yù)為“牧草之王”。然而,紫花苜蓿及其根瘤的正常生長發(fā)育經(jīng)常受到干旱、鹽、高溫、低溫等各種環(huán)境脅迫的影響。因此,利用傳統(tǒng)育種與生物技術(shù)育種手段相結(jié)合的方式培育苜蓿新品種十分重要。近年來,關(guān)于紫花苜蓿相關(guān)基因的克隆、功能驗證及遺傳轉(zhuǎn)化的研究進(jìn)展較快。本研究利用RACE-PCR法克隆了紫花苜蓿玉米黃質(zhì)環(huán)氧化酶基因Ms ZEP,并利用生物信息學(xué)軟件對其進(jìn)行序列分析。此外,利用染色體步移技術(shù)克隆了Ms ZEP基因啟動子序列,并對其順式作用元件進(jìn)行分析。以此為基礎(chǔ),利用qRT-PCR技術(shù)分析了MsZEP基因在干旱、低溫、高溫、ABA以及根瘤菌接種等不同處理下在不同組織中的表達(dá)模式;構(gòu)建了植物過表達(dá)載體pCAMBIA1300-MsZEP,利用農(nóng)桿菌介導(dǎo)法將其導(dǎo)入到煙草和紫花苜蓿中,成功獲得了MsZEP轉(zhuǎn)基因煙草和紫花苜蓿植株,并對轉(zhuǎn)基因煙草進(jìn)行功能研究�，F(xiàn)將主要研究結(jié)果總結(jié)如下:1.利用RACE-PCR技術(shù),從紫花苜蓿中成功獲得MsZEP基因的cDNA全長。序列分析結(jié)果表明,該基因cDNA全長2501bp,包含1992bp的開放閱讀框,編碼663個氨基酸殘基,GeneBank登錄號為KM044311。2.利用染色體步移技術(shù)進(jìn)行MsZEP基因啟動子序列擴(kuò)增,獲得995bp的啟動子序列。通過在線軟件PlantCare進(jìn)行了順式作用元件的分析,發(fā)現(xiàn)該啟動子區(qū)域包括若干光響應(yīng)元件、脅迫響應(yīng)元件、激素響應(yīng)元件以及TATA盒子和CAAT盒子。3.qRT-PCR分析MsZEP基因的表達(dá)模式發(fā)現(xiàn):MsZEP基因在紫花苜蓿所有組織中都有表達(dá),但主要在綠色組織(葉片和莖)中表達(dá);在不同的脅迫處理后,不同組織中MsZEP的表達(dá)模式不同。在莖葉中,干旱、低溫、高溫及ABA處理等都會使MsZEP的表達(dá)量下調(diào);而在根中其表達(dá)量的變化比較復(fù)雜。此外,根瘤菌共生能夠誘導(dǎo)MsZEP基因的表達(dá)。4.成功構(gòu)建了過表達(dá)載體pCAMBIA1300-MsZEP、瞬時表達(dá)載體MsZEP-GFP和RNAi干擾載體MsZEP-RNAi;利用瞬時表達(dá)載體MsZEP-GFP進(jìn)行煙草亞細(xì)胞定位研究表明:MsZEP基因產(chǎn)物定位于葉綠體中。5.利用農(nóng)桿菌介導(dǎo)法將MsZEP基因其轉(zhuǎn)入到煙草和紫花苜蓿中,經(jīng)組織培養(yǎng)、抗生素篩選及PCR檢測,成功獲得MsZEP轉(zhuǎn)基因煙草和紫花苜蓿。6.對MsZEP轉(zhuǎn)基因煙草進(jìn)行抗旱耐鹽性能的研究發(fā)現(xiàn):MsZEP通過改變轉(zhuǎn)基因煙草生理生化變化、提高ABA含量、調(diào)節(jié)氣孔導(dǎo)度及氣孔開度、調(diào)控內(nèi)源脅迫應(yīng)答基因表達(dá)等方式提高耐鹽性和抗旱性;7.對MsZEP轉(zhuǎn)基因煙草種子萌發(fā)試驗研究發(fā)現(xiàn):MsZEP基因通過調(diào)節(jié)轉(zhuǎn)基因煙草種子中ABA合成基因及發(fā)芽相關(guān)基因的表達(dá),促進(jìn)ABA的積累,延遲了轉(zhuǎn)基因煙草種子的萌發(fā)。8.對MsZEP轉(zhuǎn)基因煙草進(jìn)行弱光處理后生長和光合特性的研究發(fā)現(xiàn):MsZEP轉(zhuǎn)基因煙草在弱光處理后細(xì)胞排列更加緊密,葉片鮮重、葉片數(shù)目和葉面積增加,氣孔數(shù)目增多,而且其具有更強(qiáng)光合能力,生長受抑制程度遠(yuǎn)遠(yuǎn)小于野生型煙草。
[Abstract]:Medicago sativa L. (alfalfa) is a high quality leguminous pasture which can be used in root nodule biological nitrogen fixation. It has the advantages of wide cultivation, strong adaptability, good quality and high yield. However, the normal growth and development of alfalfa and its root nodules are often subjected to various environmental stresses, such as drought, salt, high temperature, low temperature and so on. Therefore, it is very important to cultivate new alfalfa varieties with the combination of traditional breeding and biotechnology breeding. In recent years, the research on the cloning, functional verification and genetic transformation of Alfalfa related genes is progressing rapidly. This study cloned Ms ZEP of Alfalfa maize yellow plasma ring oxidase gene by RACE-PCR method. In addition, the Ms ZEP gene promoter sequence was cloned by chromosome step technique and its cis acting element was analyzed. Based on this, the qRT-PCR technique was used to analyze the MsZEP gene in different groups under different treatments, such as drought, low temperature, high temperature, ABA and inoculation of Rhizobium. The expression pattern in the fabric was constructed, the plant overexpression vector pCAMBIA1300-MsZEP was constructed, and the transgenic tobacco and alfalfa were introduced into the tobacco and alfalfa by Agrobacterium tumefaciens mediated method. The MsZEP transgenic tobacco and alfalfa plants were successfully obtained, and the function of the transgenic tobacco was studied. The main research results are summarized as follows: 1. using the RACE-PCR technology, from the purple. The whole length of cDNA of MsZEP gene was successfully obtained in alfalfa. The sequence analysis showed that the gene cDNA was full of 2501bp, including 1992bp open reading frame, encoding 663 amino acid residues, GeneBank login number of KM044311.2. using chromosome step technique to amplify the promoter sequence of MsZEP gene, and obtain the promoter sequence of 995bp. The software PlantCare carried out the analysis of cis acting elements, and found that the promoter region includes a number of light response elements, stress response elements, hormone response elements and the TATA box and CAAT box.3.qRT-PCR analysis of the MsZEP gene expression pattern found that the MsZEP gene is expressed in all alfalfa tissues, but mainly in green tissue ( The expression patterns of MsZEP in different tissues were different after different stress treatments. In the stem and leaves, drought, low temperature, high temperature and ABA treatment were all down regulated, and the expression of MsZEP in the root was more complex. Moreover, the co generation of Rhizobium could induce the expression of the MsZEP gene.4. to construct the over table successfully. The vector pCAMBIA1300-MsZEP, transient expression vector MsZEP-GFP and RNAi interference carrier MsZEP-RNAi, the study on subcellular localization of tobacco using instantaneous expression vector MsZEP-GFP shows that the MsZEP gene product is located in the chloroplast by.5. using Agrobacterium mediated transformation of MsZEP gene into tobacco and alfalfa, tissue culture, antibiotics Screening and PCR detection, the study of MsZEP transgenic tobacco and alfalfa.6. on the resistance to drought and salt tolerance of MsZEP transgenic tobacco found that MsZEP can improve the salt tolerance and resistance by changing the physiological and biochemical changes of transgenic tobacco, improving the content of ABA, regulating the stomatal conductance and stomatal opening, regulating the expression of endogenous stress response genes and so on. 7. MsZEP transgenic tobacco seed germination test showed that the MsZEP gene could promote the accumulation of ABA by regulating the expression of ABA synthetic gene and the related genes in the transgenic tobacco seeds, and delayed the study of the growth and photosynthetic characteristics of the MsZEP transgenic tobacco after the weak light treatment of transgenic tobacco seeds: M After weak light treatment, sZEP transgenic tobacco had more compact cells, fresh leaf weight, increased leaf number and leaf area, increased number of stomata, and more photosynthetic capacity. The growth inhibition was much less than that of wild type tobacco.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S541.9
本文編號：2169397
[Abstract]:Medicago sativa L. (alfalfa) is a high quality leguminous pasture which can be used in root nodule biological nitrogen fixation. It has the advantages of wide cultivation, strong adaptability, good quality and high yield. However, the normal growth and development of alfalfa and its root nodules are often subjected to various environmental stresses, such as drought, salt, high temperature, low temperature and so on. Therefore, it is very important to cultivate new alfalfa varieties with the combination of traditional breeding and biotechnology breeding. In recent years, the research on the cloning, functional verification and genetic transformation of Alfalfa related genes is progressing rapidly. This study cloned Ms ZEP of Alfalfa maize yellow plasma ring oxidase gene by RACE-PCR method. In addition, the Ms ZEP gene promoter sequence was cloned by chromosome step technique and its cis acting element was analyzed. Based on this, the qRT-PCR technique was used to analyze the MsZEP gene in different groups under different treatments, such as drought, low temperature, high temperature, ABA and inoculation of Rhizobium. The expression pattern in the fabric was constructed, the plant overexpression vector pCAMBIA1300-MsZEP was constructed, and the transgenic tobacco and alfalfa were introduced into the tobacco and alfalfa by Agrobacterium tumefaciens mediated method. The MsZEP transgenic tobacco and alfalfa plants were successfully obtained, and the function of the transgenic tobacco was studied. The main research results are summarized as follows: 1. using the RACE-PCR technology, from the purple. The whole length of cDNA of MsZEP gene was successfully obtained in alfalfa. The sequence analysis showed that the gene cDNA was full of 2501bp, including 1992bp open reading frame, encoding 663 amino acid residues, GeneBank login number of KM044311.2. using chromosome step technique to amplify the promoter sequence of MsZEP gene, and obtain the promoter sequence of 995bp. The software PlantCare carried out the analysis of cis acting elements, and found that the promoter region includes a number of light response elements, stress response elements, hormone response elements and the TATA box and CAAT box.3.qRT-PCR analysis of the MsZEP gene expression pattern found that the MsZEP gene is expressed in all alfalfa tissues, but mainly in green tissue ( The expression patterns of MsZEP in different tissues were different after different stress treatments. In the stem and leaves, drought, low temperature, high temperature and ABA treatment were all down regulated, and the expression of MsZEP in the root was more complex. Moreover, the co generation of Rhizobium could induce the expression of the MsZEP gene.4. to construct the over table successfully. The vector pCAMBIA1300-MsZEP, transient expression vector MsZEP-GFP and RNAi interference carrier MsZEP-RNAi, the study on subcellular localization of tobacco using instantaneous expression vector MsZEP-GFP shows that the MsZEP gene product is located in the chloroplast by.5. using Agrobacterium mediated transformation of MsZEP gene into tobacco and alfalfa, tissue culture, antibiotics Screening and PCR detection, the study of MsZEP transgenic tobacco and alfalfa.6. on the resistance to drought and salt tolerance of MsZEP transgenic tobacco found that MsZEP can improve the salt tolerance and resistance by changing the physiological and biochemical changes of transgenic tobacco, improving the content of ABA, regulating the stomatal conductance and stomatal opening, regulating the expression of endogenous stress response genes and so on. 7. MsZEP transgenic tobacco seed germination test showed that the MsZEP gene could promote the accumulation of ABA by regulating the expression of ABA synthetic gene and the related genes in the transgenic tobacco seeds, and delayed the study of the growth and photosynthetic characteristics of the MsZEP transgenic tobacco after the weak light treatment of transgenic tobacco seeds: M After weak light treatment, sZEP transgenic tobacco had more compact cells, fresh leaf weight, increased leaf number and leaf area, increased number of stomata, and more photosynthetic capacity. The growth inhibition was much less than that of wild type tobacco.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S541.9
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相關(guān)博士學(xué)位論文 前1條
1 張志強(qiáng);紫花苜蓿玉米黃質(zhì)環(huán)氧化酶基因（MsZEP）的克隆及功能研究[D];西北農(nóng)林科技大學(xué);2016年
，本文編號：2169397
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