【摘要】：紫花苜蓿是一種多年生的豆科牧草,具有較高的營養(yǎng)價值,但土壤的鹽漬化嚴重制約了紫花苜蓿的產量。采用常規(guī)育種方法對它們進行種質改良不僅周期長而且成效低。隨著現(xiàn)代分子生物學技術的發(fā)展,利用基因工程技術改良紫花苜蓿耐鹽性成為紫花苜蓿改良育種的主要方式。本文在高效的紫花苜蓿再生體系的基礎上,以子葉節(jié)為外植體,通過農桿菌介導法將擬南芥基因SOS1-SOS2-SOS3導入到紫花苜蓿“阿爾岡金”中,經(jīng)篩選獲得了轉基因陽性植株,并對其進行抗逆性鑒定。主要研究結果如下:(1)經(jīng)PCR檢測、除草劑抗性篩選和RT-PCR鑒定,證明外源基因已整合到紫花苜蓿的基因組中,共獲得12株轉基因陽性植株,陽性率為80%。(2)以轉基因和野生型植株為材料進行盆栽耐鹽性鑒定,分別用100、200和300 mmol/L的NaCl溶液處理植株,脅迫6 d后,進行耐鹽指標的測定,結果如下:轉基因和野生型植株之間的表型存在明顯差異,即野生型植株葉片明顯變黃,并且出現(xiàn)萎蔫和枯萎跡象。在不同鹽濃度處理下,所有植株的株高均有所增長,但在100和200 mmol/L的NaCl處理下,轉基因植株的長勢顯著高于野生型植株。測定鹽脅迫下各植株的生理生化指標。結果表明:隨著處理時間的增加,所有植株的葉綠素含量均呈先上升后下降的趨勢,且野生型植株葉綠素含量均低于轉基因植株;在100和200 mmol/L的NaCl處理下,轉基因植株的細胞膜透性、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為明顯。通過測定處理前后轉基因和野生型植株根系中的Na+和K+含量。結果表明:處理后,轉基因植株根系中Na+的積累比野生型植株少,而K+的吸收多于野生型植株。說明:當在100和200 mmol/L的NaCl處理下,轉基因植株發(fā)揮SOS途徑的作用,促進Na+外排,減輕了 Na+離子對植物細胞的毒害,提高了紫花苜蓿的耐鹽性。而在300 mmol/L的NaCl處理下,轉基因和野生型植株的耐鹽指標差異不明顯。(3)以轉基因和野生型植株為材料進行水培抗旱性鑒定。分別用5%、10%和20%的PEG 6000處理植株,脅迫5 d后,進行抗旱指標的測定。結果如下:在5%和10%PEG濃度脅迫下,各植株的SOD、POD活性和葉綠素、可溶性糖含量均呈現(xiàn)下降趨勢,且野生型植株下降更為明顯;各植株的CAT活性均增加,且轉基因植株的CAT活性增加顯著;轉基因植株中的MDA和Pro的增加量均低于野生型植株。說明:在5%和10%的PEG脅迫下,轉基因植株的生理指標變化明顯,抗旱性有所提高,而在20%PEG脅迫下,二者差異不大。
[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.
【學位授予單位】：寧夏大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S541.9;Q943.2

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