SOS基因在紫花苜蓿中的表达及其抗逆性研究

发布时间：2018-09-04 10:09

【摘要】：紫花苜蓿是一种多年生的豆科牧草,具有较高的营养价值,但土壤的盐渍化严重制约了紫花苜蓿的产量。采用常规育种方法对它们进行种质改良不仅周期长而且成效低。随着现代分子生物学技术的发展,利用基因工程技术改良紫花苜蓿耐盐性成为紫花苜蓿改良育种的主要方式。本文在高效的紫花苜蓿再生体系的基础上,以子叶节为外植体,通过农杆菌介导法将拟南芥基因SOS1-SOS2-SOS3导入到紫花苜蓿“阿尔冈金”中,经筛选获得了转基因阳性植株,并对其进行抗逆性鉴定。主要研究结果如下:(1)经PCR检测、除草剂抗性筛选和RT-PCR鉴定,证明外源基因已整合到紫花苜蓿的基因组中,共获得12株转基因阳性植株,阳性率为80%。(2)以转基因和野生型植株为材料进行盆栽耐盐性鉴定,分别用100、200和300 mmol/L的NaCl溶液处理植株,胁迫6 d后,进行耐盐指标的测定,结果如下:转基因和野生型植株之间的表型存在明显差异,即野生型植株叶片明显变黄,并且出现萎蔫和枯萎迹象。在不同盐浓度处理下,所有植株的株高均有所增长,但在100和200 mmol/L的NaCl处理下,转基因植株的长势显著高于野生型植株。测定盐胁迫下各植株的生理生化指标。结果表明:随着处理时间的增加,所有植株的叶绿素含量均呈先上升后下降的趋势,且野生型植株叶绿素含量均低于转基因植株;在100和200 mmol/L的NaCl处理下,转基因植株的细胞膜透性、SOD活性和Pro含量的增加量均小于野生型植株,而POD、CAT活性和可溶性糖含量的增加量均大于野生型;各植株中MDA含量均下降,且野生型植株下降的更为明显。通过测定处理前后转基因和野生型植株根系中的Na+和K+含量。结果表明:处理后,转基因植株根系中Na+的积累比野生型植株少,而K+的吸收多于野生型植株。说明:当在100和200 mmol/L的NaCl处理下,转基因植株发挥SOS途径的作用,促进Na+外排,减轻了 Na+离子对植物细胞的毒害,提高了紫花苜蓿的耐盐性。而在300 mmol/L的NaCl处理下,转基因和野生型植株的耐盐指标差异不明显。(3)以转基因和野生型植株为材料进行水培抗旱性鉴定。分别用5%、10%和20%的PEG 6000处理植株,胁迫5 d后,进行抗旱指标的测定。结果如下:在5%和10%PEG浓度胁迫下,各植株的SOD、POD活性和叶绿素、可溶性糖含量均呈现下降趋势,且野生型植株下降更为明显;各植株的CAT活性均增加,且转基因植株的CAT活性增加显著;转基因植株中的MDA和Pro的增加量均低于野生型植株。说明:在5%和10%的PEG胁迫下,转基因植株的生理指标变化明显,抗旱性有所提高,而在20%PEG胁迫下,二者差异不大。
[Abstract]:Alfalfa is a perennial leguminous forage with high nutritional value, but soil salinization seriously restricts the yield of alfalfa. Using conventional breeding methods to improve their germplasm not only has a long period but also has low effectiveness. With the development of modern molecular biology technology, using genetic engineering technology to improve alfalfa salt tolerance has become the main way of alfalfa breeding. On the basis of efficient regeneration system of alfalfa and cotyledon node as explant, Arabidopsis thaliana gene SOS1-SOS2-SOS3 was introduced into Algonquin of alfalfa by Agrobacterium tumefaciens, and transgenic positive plants were obtained by screening. And its resistance to stress was identified. The main results are as follows: (1) after PCR detection, herbicide resistance screening and RT-PCR identification, it was proved that the exogenous gene had been integrated into the genome of alfalfa, and 12 transgenic plants were obtained. The positive rate was 80. (2) transgenic and wild-type plants were used to identify the salt tolerance of potted plants. The plants were treated with 100200 and 300 mmol/L NaCl solution respectively. After 6 days of stress, the salt tolerance indexes were determined. The results were as follows: the phenotypes of transgenic and wild-type plants were significantly different, that is, the leaves of wild-type plants turned yellow, and showed signs of wilting and wilting. Under different salt concentrations, the plant height of all plants increased, but the growth of transgenic plants was significantly higher than that of wild-type plants under 100 and 200 mmol/L NaCl treatment. Physiological and biochemical indexes of plants under salt stress were measured. The results showed that the chlorophyll content of all plants increased first and then decreased with the increase of treatment time, and the chlorophyll content of wild type plants was lower than that of transgenic plants, and under 100 and 200 mmol/L NaCl treatment, the chlorophyll content of wild type plants was lower than that of transgenic plants. Sod activity and Pro content of cell membrane permeability of transgenic plants were lower than those of wild type plants, but the increase of POD,CAT activity and soluble sugar content were higher than that of wild type, and MDA content in all plants decreased. The wild-type plants decreased more obviously. The contents of Na and K in the roots of transgenic and wild type plants were determined before and after treatment. The results showed that the accumulation of Na in the roots of transgenic plants was less than that of wild-type plants, and the absorption of K was more than that of wild-type plants. The results showed that when treated with 100 and 200 mmol/L NaCl, the transgenic plants played the role of SOS pathway, promoted Na efflux, alleviated the toxicity of Na ion to plant cells, and improved the salt tolerance of alfalfa. However, there was no significant difference in salt tolerance between transgenic and wild type plants treated with 300 mmol/L NaCl. (3) the drought resistance of transgenic and wild type plants was identified by hydroponics. The plants were treated with 10% and 20% PEG 6000, respectively, and drought resistance indexes were determined after 5 days of stress. The results were as follows: under 5% and 10%PEG concentration stress, the SOD,POD activity, chlorophyll, soluble sugar content of each plant showed a downward trend, and the wild-type plant decreased more obviously, and the CAT activity of each plant increased. The CAT activity of transgenic plants increased significantly, and the increase of MDA and Pro in transgenic plants was lower than that in wild-type plants. The results showed that under the stress of 5% and 10% of PEG, the physiological indexes of transgenic plants were obviously changed, and the drought resistance of transgenic plants was improved, but under 20%PEG stress, the difference between them was not significant.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S541.9;Q943.2

【参考文献】