盐生草HgNHX1基因及其启动子的功能研究

发布时间：2018-06-27 05:50

本文选题：盐生草 + HgNHX1基因　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：盐胁迫是全球范围内重要的非生物胁迫之一,它会使植物所处的土壤环境Na~+浓度增高,从而导致植物离子稳态被破坏并发生渗透胁迫。而液泡膜Na~+/H~+逆向转运蛋白(NHX)对植物体的离子平衡、耐盐性以及植株的整个发育过程都起着很重要的作用。盐生草(Halogeton glomeratus)为藜科(Chenopodiaceae)盐生草属一年生双子叶植物,广泛分布于我国西北旱区及半干旱区,其茎、叶均高度肉质化,可以在盐碱化环境中正常生长,是挖掘植物耐盐基因及研究耐盐调控机理的良好材料,对改良作物抗逆性及培育耐盐碱作物具有重要的应用价值。其最主要的耐盐机制是将盐毒性离子区隔化在细胞液泡中。因此,可在分子水平研究其耐盐机理。基因工程中以35S启动子的使用最为广泛,它能够满足在植物组织器官中非特异性高效表达,但是这种表达方式会打破植物原有的代谢平衡,从而影响植物的正常生长。为了减少这种不利影响,克隆新的具有组织特异性的启动子在基因工程的研究与发展中意义深远。本实验以盐生草为材料克隆了HgNHX1基因上游的启动子(pHgNHX1),并对pHgNHX1的功能进行了探索;同时对HgNHX1基因转化拟南芥后进行耐盐抗旱功能研究。主要结果如下:1.根据已获得的pHgNHX1序列设计上下游引物,克隆HgNHX1基因上游的启动子,并利用软件PlantCARE和PLACE等对pHgNHX1进行功能元件的分析,发现pHgNHX1中除具有核心启动子元件外还包含响应逆境胁迫诱导和植物激素诱导的功能元件,说明该启动子具有典型启动子的一般特征。2.构建缺失HgNHX1基因上游不同长度的启动子表达载体pBI-pHgNHX1并转化烟草和拟南芥,对阳性克隆植株进行GUS组织化学染色,结果表明:4个不同长度片段的启动子都可驱使GUS基因在烟草叶片和拟南芥株系中表达,说明该启动子具有启动子活性,并且在HgNHX1基因上游611 bp得片段就包含启动子的核心表达元件,但?1染色结果较其他三个片段稍浅。3.对已成功转化了HgNHX1基因上游1523 bp启动子的烟草和拟南芥不同组织进行GUS染色,结果显示在烟草的叶和茎、拟南芥不同生长时期、不同组织中均可显色,说明该启动子具有组成型启动子的特性。4.将本实验室构建的表达载体pCAMBIA1301-HgNHX1利用浸花法转化拟南芥,种植阳性T2代植株并进行耐盐抗旱功能研究,结果显示在干旱及盐胁迫下该基因表达量虽然增加了,但是通过表型变化的观察及相关生理指标的测定发现转基因拟南芥的抗旱性和耐盐性并没有提高。
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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