矮秆波兰小麦重金属转运基因TpNRAMPs克隆和表达分析

发布时间：2018-05-15 02:19

本文选题：矮秆波兰小麦 + NRAMP　；参考：《四川农业大学》2016年硕士论文

【摘要】：自然抗性相关巨噬细胞蛋白家族(Natural resistance-associated macrophage proteins, NRAMP)是一类高度保守的跨膜蛋白,广泛存在于原生生物、真菌、植物和动物中。该家族最早在小鼠体内发现(Nramp1),控制小鼠体内寄生虫感染自然抗性。拟南芥(Arbidopsis thaliana)和水稻(Oryza stativa)等植物中的NRAMP基因参与植物体重金属吸收、转运以及再分布,维持植物体内金属平衡；同时,NRAMP家族还同植物抗性相关。至今,已在拟南芥和水稻中分别发现6个和7个NRAMP基因,理论上小麦中也应该含有NRAMP基因,然而该家族在小麦中鲜有报道。波兰小麦(2n=4x=28,AABB,Triticum polonicum)是禾本科小麦属重要的四倍体二粒栽培小麦,是普通小麦遗传改良的重要种质资源。波兰小麦可能是伊斯帕罕小麦(2n=4x=28,AABB,T.ispahanicum)与硬粒小麦(2n=4x=28,AABB,T.durum)的杂种,同普通小麦及其余四倍体小麦的遗传相似性较低。矮秆波兰小麦是在我国新疆地区发现的具有矮秆性状的波兰小麦,具有株高矮化、长穗等一系列性状。矮秆波兰小麦能积累较高含量的镉和锌,但其原理未知。NRAMP作为重金属元素转运蛋白,在植物体耐重金属胁迫方面具有重要的作用。本实验旨在分离鉴定矮秆波兰小麦中的NRAMP基因,并通过qRT-PCR初步分析矮秆波兰小麦NRAMP在重金属处理下表达模式的变化,从而丰富对植物NRAMP转运蛋白家族的认识,为小麦重金属遗传修饰奠定基础和提供依据。本研究利用矮秆波兰小麦转录组数据库,筛选获得4条编码序列完整的NRAMP序列,以此作为参考序列设计引物,从矮秆波兰小麦的根和叶中进行克隆,同时利用qRT-PCR对TpNRAMPs在7种不同重金属元素处理下基因表达情况进行了分析。主要结果如下：1.矮秆波兰小麦具有较强的耐金属属性。金属处理24小时后,矮秆波兰小麦的根和叶片均积累了大量的金属元素。其中铜含量最高,分别达到了125.88 mg/g和38.53mg/g,但同时也影响了植株的生长。此外,根和叶片也积累了高含量的镉、锌和铁等元素,但植株生长没有受到影响,表明矮秆波兰小麦具有一定的耐金属属性。2.克隆获得5条矮秆波兰小麦TpNRAMPs基因序列,分别命名为TpNRAMP2.1、 TpNRAMP2.2、TpNRAMP3、TpNRAMP5和TpNRAMP6。同时,小麦基因组序列信息比对获得了普通小麦的TaNRAMP1、TaNRAMP7及一未命名的TaNRAMP基因,从而确定小麦具有7个或7个以上NRAMP基因。3.在不同金属胁迫下,TpNRAMP2.1、TpNRAMP3、TpNRAMP5和TpNRAMP6的表达模式变化不同,表现出不同的金属敏感性,可能具有不同的金属吸收和转运的能力。TpNRAMP2.1在矮秆波兰小麦的根部对镁、铁和铜最敏感；在叶片中,对镁和锌最敏感。其次,TpNRAMP3在根部对铁、铜和镉最敏感且其表达量均显著提高；在叶片中,TpNRAMP3对镉无反应。第三,TpNRAMP5在根部对铁和锌最敏感；在叶片中,对镉的敏感性最高。最后,TpNRAMP6在根部对铁最敏感,而对铅无反应：在叶片中,对镁和锌最敏感。同时,这4个基因在矮秆波兰小麦的根部对铁均表现出最强的敏感性。
[Abstract]:The natural resistance related macrophage protein family (Natural resistance-associated macrophage proteins (NRAMP)) is a highly conserved type of transmembrane protein, which is widely found in protists, fungi, plants and animals. The family was first found in mice (Nramp1) to control the natural resistance of the parasites in mice. Arabidopsis (Arbidop). The NRAMP genes of SIS thaliana) and rice (Oryza stativa) are involved in plant body weight metal absorption, transport and redistribution to maintain the balance of metal in plants. At the same time, the NRAMP family is also related to plant resistance. Up to now, 6 and 7 NRAMP genes have been found in Arabidopsis and rice respectively. In theory, NRAMP should also contain NRAMP. Gene, however, this family is rarely reported in wheat. Poland wheat (2n=4x=28, AABB, Triticum polonicum) is an important tetraploid two grain wheat cultivated in the genus grasses. It is an important germplasm resource for genetic improvement of common wheat. Poland wheat may be 2n=4x=28, AABB, T.ispahanicum, and hard grain wheat (2n=4x=28, AABB, T). The genetic similarity of.Durum) was lower than that of common wheat and other four triploid wheat. Dwarf Poland wheat was found in Xinjiang region of China with dwarfing character of Poland wheat, which had a series of characters such as dwarfing plant height and long panicle. The dwarf Poland wheat could accumulate high content of cadmium and zinc, but the principle of.NRAMP was not known as heavy gold. The purpose of this experiment is to isolate and identify the NRAMP gene in the dwarf Poland wheat, and to analyze the changes in the expression pattern of the dwarf Poland wheat NRAMP under the heavy metal treatment by qRT-PCR, so as to enrich the understanding of the plant NRAMP transporter family. This study uses the Poland wheat transcriptional database of dwarf Poland wheat transcriptional group to screen and obtain 4 complete sequences of coded sequences, which are designed as primers for reference sequences, cloned from the roots and leaves of dwarf Poland wheat, and at the same time using qRT-PCR to TpNRAMPs in 7 different heavy metal elements The main results were as follows: 1. the dwarf Poland wheat had strong metal resistance. After 24 hours of metal treatment, the roots and leaves of the dwarf Poland wheat accumulated a large number of metal elements, of which the highest copper content reached 125.88 mg/g and 38.53mg/g respectively, but it also affected the plant. In addition, the roots and leaves also accumulated a high content of cadmium, zinc and iron, but the growth of the plant was not affected. It showed that the dwarf Poland wheat had a certain metal resistance.2. clone to obtain 5 dwarf Poland wheat TpNRAMPs gene sequences, named TpNRAMP2.1, TpNRAMP2.2, TpNRAMP3, TpNRAMP5 and TpNRAMP6., respectively. Genomic sequence information comparison obtained the TaNRAMP1, TaNRAMP7 and unnamed TaNRAMP gene of common wheat, thus determining that the expression patterns of TpNRAMP2.1, TpNRAMP3, TpNRAMP5 and TpNRAMP6 in Wheat with 7 or more than 7 NRAMP genes were different under different metal stresses, showing different metal sensitivities and may be different. The ability of.TpNRAMP2.1 to absorb and transport metals is most sensitive to magnesium, iron and copper at the roots of the dwarf Poland wheat; in the leaves, it is most sensitive to magnesium and zinc. Secondly, TpNRAMP3 is most sensitive to iron, copper and cadmium in the root and its expression is significantly increased; in the leaves, TpNRAMP3 does not respond to cadmium. Third, TpNRAMP5 is most sensitive to iron and zinc at the root. The sensitivity to cadmium is the highest in the leaves. Finally, TpNRAMP6 is most sensitive to iron at the root and does not respond to lead: in leaves, it is most sensitive to magnesium and zinc. At the same time, these 4 genes are most sensitive to iron in the roots of dwarf Poland wheat.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1

【相似文献】