转蜡样芽孢杆菌acdS基因生菜幼苗耐盐性的提高

发布时间：2018-06-19 07:20

本文选题：转基因生菜 + acdS基因　；参考：《郑州大学》2017年硕士论文

【摘要】：全球有近10%的耕地属于盐碱地,土壤盐渍化是影响农业生产和限制农作物产量的重要因素之一,严重影响耕地的有效利用,影响了农业的可持续发展。生菜(Lactuca)以其营养价值高,低热量,可生食而成为人们所喜爱的重要蔬菜作物。生菜在栽培过程中经常因为干旱、盐渍、高温的影响,导致品质和产量下降。利用基因工程进行种质创新和品种改良成为提高作物耐盐性的一个快速有效的方法,建立稳定高效的生菜转化体系,培育耐盐生菜新品种,为盐碱地的生菜及作物种植提供技术支撑和理论依据。ACC脱氨酶可以将乙烯的合成前体ACC(1-氨基环丙烷-1羧酸)水解成氨和α-酮丁酸,从而减少植物中产生的乙烯量,以提高植物抗性。论文克隆了蜡样芽孢杆菌(HK)编码ACC脱氨酶的acd S基因,构建植物表达载体35S::acd S-GFP,并利用农杆菌介导的叶盘法转化香港玻璃生菜。本实验优化了香港玻璃生菜的遗传转化体系。先将生菜叶片在分化培养基上预培养2d,其中分化培养基为MS+6-BA0.5mg/L+NAA0.1mg/L,然后在OD600为0.6的农杆菌菌液中浸泡8min进行转化,转化后共培养3d,移入分化筛选培养基,筛选抗生素选择Hyg10mg/L,抑菌抗生素选择Cef300mg/L,选用1/2MS+0.05mg/L NAA+Hyg10mg/L作为生根培养基。经融合基因PCR分析和Western blot蛋白表达鉴定,融合基因成功整合到生菜基因组中,获得了转蜡样芽孢杆菌acd S基因稳定表达的转基因生菜幼苗株系,融合基因定位在根系细胞的细胞膜上。外源acd S基因的导入使转基因生菜表现出明显的ACC脱氨酶活性。转基因生菜在盐胁迫实验结果表明,转基因生菜在盐胁迫下的生长优于野生型,在最高盐处理时优势更明显。转基因生菜中脯氨酸含量在300mmol/LNa Cl时达到802.02μg/g,是野生型的1.36倍;在300mmol/L Na Cl时,转基因生菜中可溶性糖含量为43033.80μg/g,是野生型的2.09倍,野生型植株为20533.80μg/g;在Na Cl 300mmol/L时,野生型生菜SOD活性为不加盐对照组的2.14倍,转基因生菜SOD活性为不加盐对照组的3.47倍,活性显著高于野生型植株;在Na Cl200mmol/L时,转基因生菜中叶绿素含量为0.16mg/g,非转基因生菜叶绿素含量为0.09mg/g;在Na Cl 300mmol/L时,转基因生菜叶片相对含水量为70.95%,而野生型相对含水量为60.95%;在Na Cl 300mmol/L时,野生型植株的MDA含量为4.80×10-3μmol/g,转基因生菜的MDA含量为4.00×10-3μmol/g,显著低于WT植株(P0.05)。综上所述,导入acd S基因可有效消除盐胁迫对生菜的损害,提高了转基因生菜的耐盐性。本实验中,蜡样芽孢杆菌的ACC脱氨酶基因被导入生菜,获得ACC脱氨酶含量显著增加的转基因生菜株系,并通过模拟盐胁迫研究导入acd S基因对转基因生菜耐盐性的影响,为培育耐盐生菜品种奠定了基础。
[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 伪 -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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S636.2

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