本文選題：OsNHX1基因 + 農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化��； 參考：《吉林大學(xué)》2016年碩士論文

【摘要】：土地鹽堿化是現(xiàn)代農(nóng)業(yè)面臨的重要問題之一,為了增加土地利用率,提高農(nóng)作物產(chǎn)量,研制具有高耐鹽性的農(nóng)作物具有十分重要的意義。本研究通過從水稻中克隆耐鹽基因OsNHX1,并利用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法將其轉(zhuǎn)入番茄中,以期培育出具有高耐鹽性的轉(zhuǎn)基因番茄。NHX基因是與抗旱耐鹽相關(guān)的重要基因之一,NHX基因控制表達Na+(K+)/H+逆向轉(zhuǎn)運蛋白,它能夠促進鈉離子在液泡中積累,并且調(diào)節(jié)細胞質(zhì)pH,使植株獲得耐鹽能力。番茄是茄科、番茄屬一至兩年生草本植物,栽培面積廣大。番茄中含有糖類、礦物質(zhì)、番茄紅素等多種成分,具有健胃消食、涼血平肝、清熱解毒等功效。由于番茄自花授粉、染色體圖譜清楚、后代多、遺傳轉(zhuǎn)化操作容易、較易栽培等優(yōu)點,成為生物學(xué)研究最多的模式植物之一。本研究的結(jié)果如下:(1)含有OsNHX1基因的農(nóng)桿菌的對數(shù)生長期為10~15小時,選用10~15小時的農(nóng)桿菌進行轉(zhuǎn)化實驗;(2)篩選出番茄再生培養(yǎng)基發(fā)芽培養(yǎng)基:1/2MS固體培養(yǎng)基;誘導(dǎo)培養(yǎng)基:MS固體培養(yǎng)基(3%蔗糖)+0.2mg/L IAA+2mg/L 6-BA;共培養(yǎng)培養(yǎng)基:MS固體培養(yǎng)基(3%蔗糖)+0.2mg/L IAA+2mg/L6-BA+0.2mg/L AS;生根培養(yǎng)基:MS固體培養(yǎng)基(3%蔗糖)+0.1mg/L NAA+100mg/L Cef(3)轉(zhuǎn)化條件的確定OD600=3.0時的農(nóng)桿菌重懸液,重懸液成分為MS(3%蔗糖)+0.2mg/L IAA+2mg/L 6-BA,預(yù)培養(yǎng)2天,侵染8分鐘,共培養(yǎng)四天。(4)抗性植株獲得及檢測通過對番茄外植體進行轉(zhuǎn)化,共獲得抗性芽190個;生根煉苗移栽后獲得抗性植株28株。經(jīng)過PCR檢測初步證實OsNHX1基因整合入番茄基因組中;經(jīng)Southernblot雜交檢測進一步證實目的基因以不同拷貝的形式整合到番茄基因組的不同位點中;經(jīng)RT-PCR檢測證實,抗性植株中OsNHX1基因在RNA水平表達。(5)抗性植株耐鹽性檢測通過對植株不同生長時期施加鹽脅迫,檢測抗性植株相比野生型耐鹽性是否有提高,結(jié)果顯示種子萌發(fā)率與野生型相比無顯著差別,但平均萌發(fā)時間比野生型早2天;幼苗生長情況與野生型相比較好,不但從感受脅迫至出現(xiàn)枯萎的時間比野生型長,最終存活率也比野生型高。說明實驗中轉(zhuǎn)入的OsNHX1基因提高了番茄植株的耐鹽性。本研究建立了番茄再生及轉(zhuǎn)化體系,并將取自水稻的OsNHX1基因成功轉(zhuǎn)入番茄中進行表達,提高了植株的耐鹽性,為培育具有高耐鹽性農(nóng)作物打下了理論基礎(chǔ)。但有關(guān)植物耐鹽機理的研究尚未透徹,也有許多有關(guān)植物耐鹽的基因還沒有被克隆,因此,通過轉(zhuǎn)基因的方式提高植物耐鹽性仍需進行更深入的研究。
[Abstract]:Land salinization is one of the most important problems faced by modern agriculture. In order to increase land use efficiency and increase crop yield, it is of great significance to develop crops with high salt tolerance. In this study, the salt-tolerant gene OsNHX1 was cloned from rice and transferred into tomato by Agrobacterium tumefaciens mediated genetic transformation. The aim of this study is to develop transgenic tomato. NHX gene with high salt tolerance, which is one of the important genes related to drought resistance and salt tolerance. The NHX gene controls the expression of Na ~ + K ~ + / H antiporter, which can promote the accumulation of sodium ions in vacuoles. And adjust the pH of the cytoplasm to make the plant gain salt tolerance ability. Tomato is the family of eggplant, tomato is one to two years old herbaceous plant, cultivated area is broad. Tomato contains sugars, minerals, lycopene and other ingredients, with stomach digestion, cooling blood and liver, clearing heat and detoxification. Because of its advantages such as self-pollination, clear chromosome map, multiple progeny, easy genetic transformation and easy cultivation, tomato has become one of the most studied model plants in biology. The results of this study were as follows: (1) the logarithmic growth period of Agrobacterium tumefaciens containing OsNHX1 gene was 10 ~ 15 hours. Agrobacterium tumefaciens with 10 ~ 15 hours was selected for transformation experiment. Inducing medium MS solid medium with 3% sucrose) 0.2 mg / L IAA 2 mg / L 6-BA; Co-culture medium 1% MS solid medium with 3% sucrose) 0.2 mg / L IAA 2 mg / L 6-BA 0.2 mg / L AS; rooting medium: MS solid medium with 3% sucrose) 0.1 mg / L NAA 100 mg / L Cef3) heavy suspension of Agrobacterium tumefaciens at OD600g 3.0. The composition of the heavy suspension was MSN 3% sucrose) 0.2 mg / L IAA 2 mg / L 6-BA, pre-cultured for 2 days, infected for 8 minutes, and co-cultured for 4 days. The resistant plants were obtained and detected by transformation of tomato explants, a total of 190 resistant buds were obtained. 28 resistant plants were obtained after rooting and transplanting. The integration of OsNHX1 gene into tomato genome was preliminarily confirmed by PCR, further confirmed by Southern blot hybridization, and the integration of OsNHX1 gene into different loci of tomato genome was further confirmed by Southern blot hybridization. Expression of OsNHX1 gene in resistant plants at RNA level.) Salt tolerance of resistant plants was tested by applying salt stress at different growth stages to determine whether the resistance plants were more salt tolerant than wild type plants. The results showed that there was no significant difference between the seed germination rate and the wild type, but the average germination time was 2 days earlier than that of the wild type, and the seedling growth was better than that of the wild type. The final survival rate was also higher than that of the wild type. The results showed that the OsNHX1 gene transferred in the experiment improved the salt tolerance of tomato plants. In this study, the tomato regeneration and transformation system was established, and the OsNHX1 gene from rice was successfully transferred to tomato for expression, which improved the salt tolerance of the plants and laid a theoretical foundation for the cultivation of crops with high salt tolerance. However, the mechanism of salt tolerance in plants has not been thoroughly studied, and many genes related to salt tolerance in plants have not been cloned. Therefore, it is necessary to further study how to improve the salt tolerance of plants by transgenic methods.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2;S641.2

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