本文選題：轉基因生菜 + acdS基因　； 參考：《鄭州大學》2017年碩士論文

【摘要】：全球有近10%的耕地屬于鹽堿地,土壤鹽漬化是影響農業(yè)生產和限制農作物產量的重要因素之一,嚴重影響耕地的有效利用,影響了農業(yè)的可持續(xù)發(fā)展。生菜(Lactuca)以其營養(yǎng)價值高,低熱量,可生食而成為人們所喜愛的重要蔬菜作物。生菜在栽培過程中經常因為干旱、鹽漬、高溫的影響,導致品質和產量下降。利用基因工程進行種質創(chuàng)新和品種改良成為提高作物耐鹽性的一個快速有效的方法,建立穩(wěn)定高效的生菜轉化體系,培育耐鹽生菜新品種,為鹽堿地的生菜及作物種植提供技術支撐和理論依據(jù)。ACC脫氨酶可以將乙烯的合成前體ACC(1-氨基環(huán)丙烷-1羧酸)水解成氨和α-酮丁酸,從而減少植物中產生的乙烯量,以提高植物抗性。論文克隆了蠟樣芽孢桿菌(HK)編碼ACC脫氨酶的acd S基因,構建植物表達載體35S::acd S-GFP,并利用農桿菌介導的葉盤法轉化香港玻璃生菜。本實驗優(yōu)化了香港玻璃生菜的遺傳轉化體系。先將生菜葉片在分化培養(yǎng)基上預培養(yǎng)2d,其中分化培養(yǎng)基為MS+6-BA0.5mg/L+NAA0.1mg/L,然后在OD600為0.6的農桿菌菌液中浸泡8min進行轉化,轉化后共培養(yǎng)3d,移入分化篩選培養(yǎng)基,篩選抗生素選擇Hyg10mg/L,抑菌抗生素選擇Cef300mg/L,選用1/2MS+0.05mg/L NAA+Hyg10mg/L作為生根培養(yǎng)基。經融合基因PCR分析和Western blot蛋白表達鑒定,融合基因成功整合到生菜基因組中,獲得了轉蠟樣芽孢桿菌acd S基因穩(wěn)定表達的轉基因生菜幼苗株系,融合基因定位在根系細胞的細胞膜上。外源acd S基因的導入使轉基因生菜表現(xiàn)出明顯的ACC脫氨酶活性。轉基因生菜在鹽脅迫實驗結果表明,轉基因生菜在鹽脅迫下的生長優(yōu)于野生型,在最高鹽處理時優(yōu)勢更明顯。轉基因生菜中脯氨酸含量在300mmol/LNa Cl時達到802.02μg/g,是野生型的1.36倍;在300mmol/L Na Cl時,轉基因生菜中可溶性糖含量為43033.80μg/g,是野生型的2.09倍,野生型植株為20533.80μg/g;在Na Cl 300mmol/L時,野生型生菜SOD活性為不加鹽對照組的2.14倍,轉基因生菜SOD活性為不加鹽對照組的3.47倍,活性顯著高于野生型植株;在Na Cl200mmol/L時,轉基因生菜中葉綠素含量為0.16mg/g,非轉基因生菜葉綠素含量為0.09mg/g;在Na Cl 300mmol/L時,轉基因生菜葉片相對含水量為70.95%,而野生型相對含水量為60.95%;在Na Cl 300mmol/L時,野生型植株的MDA含量為4.80×10-3μmol/g,轉基因生菜的MDA含量為4.00×10-3μmol/g,顯著低于WT植株(P0.05)。綜上所述,導入acd S基因可有效消除鹽脅迫對生菜的損害,提高了轉基因生菜的耐鹽性。本實驗中,蠟樣芽孢桿菌的ACC脫氨酶基因被導入生菜,獲得ACC脫氨酶含量顯著增加的轉基因生菜株系,并通過模擬鹽脅迫研究導入acd S基因對轉基因生菜耐鹽性的影響,為培育耐鹽生菜品種奠定了基礎。
[Abstract]:Soil salinization is one of the important factors affecting agricultural production and limiting crop yield, which seriously affects the effective utilization of cultivated land and the sustainable development of agriculture. Lettuce (Lactuca) has become an important vegetable crop for its high nutritional value, low calorie and raw food. The quality and yield of lettuce are often reduced due to drought, salinity and high temperature. Genetic engineering for germplasm innovation and variety improvement has become a rapid and effective method to improve the salt-tolerance of crops, to establish a stable and efficient transformation system of lettuce, and to cultivate new salt-tolerant lettuce varieties. To provide technical support and theoretical basis for lettuce and crop cultivation in saline-alkali soil. ACC deaminase can hydrolyze the precursor of ethylene synthesis, ACC-1-aminocyclopropane--1 carboxylic acid, to ammonia and 偽 -ketobutyric acid, thus reducing the amount of ethylene produced in plants. To improve plant resistance. In this paper, the acd S gene encoding ACC deaminase was cloned from Bacillus cereus, and the plant expression vector 35S: ACD S-GFP was constructed and transformed into Hong Kong vitreous lettuce by Agrobacterium tumefaciens mediated leaf disk method. The genetic transformation system of Hong Kong lettuce was optimized. The leaves were precultured on the differentiation medium for 2 days, in which the differentiation medium was MS 6-BA 0.5 mg / L NAA 0.1 mg / L, then soaked in Agrobacterium tumefaciens solution with OD600 0.6 for 8min transformation, and then cultured for 3 days, then transferred to the differentiation screening medium. Hyg 10 mg / L, Cef300 mg / L, 1 / 2 MS 0.05 mg / L NAA Hyg 10 mg / L as rooting medium. The fusion gene was successfully integrated into the genome of lettuce by PCR analysis and Western blot protein expression. The transgenic lettuce seedling lines with stable expression of acd S gene of Bacillus cereus were obtained. The fusion gene was located on the cell membrane of root cells. The introduction of exogenous acd S gene made the transgenic lettuce show obvious activity of acd deaminase. The results of salt stress showed that transgenic lettuce grew better than wild type under salt stress, and was more dominant in the highest salt treatment. The proline content in transgenic lettuce was 802.02 渭 g / g at 300 mmol / L NaCl, 1.36 times that of wild type; at 300 mmol / L NaCl, the soluble sugar content of transgenic lettuce was 43033.80 渭 g / g, 2.09 times that of wild type and 20533.80 渭 g / g of wild type; at NaCl 300 mmol / L, The SOD activity of wild lettuce and transgenic lettuce was 2.14 times and 3.47 times as much as that of control group, which was significantly higher than that of wild type plant, and the SOD activity of transgenic lettuce was 3.47 times higher than that of wild type plant, and the SOD activity of transgenic lettuce was significantly higher than that of wild type plant. The chlorophyll content of transgenic lettuce is 0.16 mg / g, and that of non-transgenic lettuce is 0.09 mg / g; at NaCl 300 mmol / L, the relative water content of transgenic vegetable leaf is 70.95%, while that of wild type is 60.95%; at NaCl 300 mmol / L, The MDA content of wild type plants was 4.80 脳 10 -3 渭 mol / g, and that of transgenic lettuce was 4.00 脳 10 -3 渭 mol / g, which was significantly lower than that of WT plants. In conclusion, the introduction of acd S gene can effectively eliminate the damage of salt stress on lettuce and improve the salt tolerance of transgenic lettuce. In this experiment, the ACC deaminase gene of Bacillus cereus was introduced into lettuce, and transgenic lettuce lines with significant increase in ACC deaminase content were obtained. The effect of introducing acd S gene on salt tolerance of transgenic lettuce was studied by simulating salt stress. It laid a foundation for the cultivation of salt-tolerant lettuce varieties.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S636.2

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相關碩士學位論文 前2條

1 劉方方;轉蠟樣芽孢桿菌acdS基因生菜幼苗耐鹽性的提高[D];鄭州大學;2017年

2 劉珂;轉蠟樣芽孢桿菌acdS基因改善煙草耐鹽性研究[D];鄭州大學;2017年

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本文編號：2039083