蓖麻RcNHX1基因的功能分析

发布时间：2018-03-13 18:27

本文选题：蓖麻　切入点：盐胁迫　出处：《内蒙古民族大学》2017年硕士论文　论文类型：学位论文

【摘要】：土壤盐碱化一直是亟待解决的世界性农业难题之一,严重影响了全球农业经济的发展及生态环境的保护。利用基因工程选育耐盐作物品种,已成为利用和改良盐碱地的一条经济有效途径。近年来,随着蓖麻产业的发展,人们对蓖麻的生产和加工的重视程度越来越高,而针对蓖麻耐盐相关基因的发掘和利用的研究较少。为了挖掘耐盐相关基因,本研究以当地主推的骨干自交系通蓖5号为材料,利用RACE-PCR的方法成功克隆了蓖麻液泡膜Na~+/H~+逆向转运蛋白(vacuolar Na~+/H~+antiporter,NHX)基因RcNHX1。RcNHX1基因包括一个完整的开放阅读框,推测编码544个氨基酸。亚细胞定位试验表明RcNHX1融合蛋白主要积累于细胞膜与液泡膜。以蓖麻子叶节为外植体,构建植物过表达载体、干扰载体,利用农杆菌进行遗传转化。PCR和Southern blot检测表明共获得2个稳定遗传的过表达株系。对过表达RcNHX1转基因蓖麻在苗期抗盐表型鉴定发现:150 mmol/L NaCl处理14 d后,转基因植株的株高和干重均显著高于野生型。盐胁迫后,转基因植株脯氨酸的含量显著高于野生型,而丙二醛的含量及相对电解质渗透率显著降低。此外,相比于野生型,盐胁迫后转基因植株叶片和根中Na~+与K~+离子含量的升高为RcNHX1提高转基因植株的抗盐性提供了可能的分子机制。
[Abstract]:Soil salinization has always been one of the world agricultural problems to be solved, which has seriously affected the development of global agricultural economy and the protection of ecological environment. In recent years, with the development of castor industry, people pay more and more attention to the production and processing of castor. In order to excavate salt-tolerant genes, the local backbone inbred line Tonggri 5 was used as the material. RACE-PCR method was used to clone the RcNHX1.RcNHX1 gene of the vacuolar Na ~ / / H ~ antiporterus NH _ Xgene of castor, including a complete open reading frame. Subcellular localization test showed that RcNHX1 fusion protein was mainly accumulated in cell membrane and vacuolar membrane. Plant overexpression vector was constructed with castor cotyledon as explant. The results of Agrobacterium tumefaciens genetic transformation. PCR and Southern blot analysis showed that two stable genetic overexpression lines were obtained. After 14 days of treatment, the overexpressed RcNHX1 transgenic castor plants were identified as salt-tolerant phenotypes at seedling stage and were treated with 1: 150 mmol/L NaCl for 14 days. The plant height and dry weight of transgenic plants were significantly higher than that of wild type. After salt stress, the content of proline was significantly higher than that of wild type, while the content of malondialdehyde and the relative electrolyte permeability of transgenic plant were significantly lower than that of wild type. The increase of Na ~ and K ~ + contents in leaves and roots of transgenic plants after salt stress may provide a possible molecular mechanism for RcNHX1 to improve the salt tolerance of transgenic plants.
【学位授予单位】：内蒙古民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.6;Q943.2

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