紫花苜蓿玉米黄质环氧化酶基因（MsZEP）的克隆及功能研究
发布时间：2018-08-07 08:03
【摘要】：紫花苜蓿(Medicago sativa L.)是一种能够通过根瘤生物固氮的高品质豆科牧草,具有栽培范围广、适应性强、品质好、产量高等优点,被誉为“牧草之王”。然而,紫花苜蓿及其根瘤的正常生长发育经常受到干旱、盐、高温、低温等各种环境胁迫的影响。因此,利用传统育种与生物技术育种手段相结合的方式培育苜蓿新品种十分重要。近年来,关于紫花苜蓿相关基因的克隆、功能验证及遗传转化的研究进展较快。本研究利用RACE-PCR法克隆了紫花苜蓿玉米黄质环氧化酶基因Ms ZEP,并利用生物信息学软件对其进行序列分析。此外,利用染色体步移技术克隆了Ms ZEP基因启动子序列,并对其顺式作用元件进行分析。以此为基础,利用qRT-PCR技术分析了MsZEP基因在干旱、低温、高温、ABA以及根瘤菌接种等不同处理下在不同组织中的表达模式;构建了植物过表达载体pCAMBIA1300-MsZEP,利用农杆菌介导法将其导入到烟草和紫花苜蓿中,成功获得了MsZEP转基因烟草和紫花苜蓿植株,并对转基因烟草进行功能研究。现将主要研究结果总结如下:1.利用RACE-PCR技术,从紫花苜蓿中成功获得MsZEP基因的cDNA全长。序列分析结果表明,该基因cDNA全长2501bp,包含1992bp的开放阅读框,编码663个氨基酸残基,GeneBank登录号为KM044311。2.利用染色体步移技术进行MsZEP基因启动子序列扩增,获得995bp的启动子序列。通过在线软件PlantCare进行了顺式作用元件的分析,发现该启动子区域包括若干光响应元件、胁迫响应元件、激素响应元件以及TATA盒子和CAAT盒子。3.qRT-PCR分析MsZEP基因的表达模式发现:MsZEP基因在紫花苜蓿所有组织中都有表达,但主要在绿色组织(叶片和茎)中表达;在不同的胁迫处理后,不同组织中MsZEP的表达模式不同。在茎叶中,干旱、低温、高温及ABA处理等都会使MsZEP的表达量下调;而在根中其表达量的变化比较复杂。此外,根瘤菌共生能够诱导MsZEP基因的表达。4.成功构建了过表达载体pCAMBIA1300-MsZEP、瞬时表达载体MsZEP-GFP和RNAi干扰载体MsZEP-RNAi;利用瞬时表达载体MsZEP-GFP进行烟草亚细胞定位研究表明:MsZEP基因产物定位于叶绿体中。5.利用农杆菌介导法将MsZEP基因其转入到烟草和紫花苜蓿中,经组织培养、抗生素筛选及PCR检测,成功获得MsZEP转基因烟草和紫花苜蓿。6.对MsZEP转基因烟草进行抗旱耐盐性能的研究发现:MsZEP通过改变转基因烟草生理生化变化、提高ABA含量、调节气孔导度及气孔开度、调控内源胁迫应答基因表达等方式提高耐盐性和抗旱性;7.对MsZEP转基因烟草种子萌发试验研究发现:MsZEP基因通过调节转基因烟草种子中ABA合成基因及发芽相关基因的表达,促进ABA的积累,延迟了转基因烟草种子的萌发。8.对MsZEP转基因烟草进行弱光处理后生长和光合特性的研究发现:MsZEP转基因烟草在弱光处理后细胞排列更加紧密,叶片鲜重、叶片数目和叶面积增加,气孔数目增多,而且其具有更强光合能力,生长受抑制程度远远小于野生型烟草。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S541.9
本文编号：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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S541.9
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1 张志强;紫花苜蓿玉米黄质环氧化酶基因（MsZEP）的克隆及功能研究[D];西北农林科技大学;2016年
，本文编号：2169397
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