OsNHX1基因的克隆及其遗传转化番茄的研究

发布时间：2018-06-09 16:48

本文选题：OsNHX1基因 + 农杆菌介导的遗传转化　；参考：《吉林大学》2016年硕士论文

【摘要】：土地盐碱化是现代农业面临的重要问题之一,为了增加土地利用率,提高农作物产量,研制具有高耐盐性的农作物具有十分重要的意义。本研究通过从水稻中克隆耐盐基因OsNHX1,并利用农杆菌介导的遗传转化方法将其转入番茄中,以期培育出具有高耐盐性的转基因番茄。NHX基因是与抗旱耐盐相关的重要基因之一,NHX基因控制表达Na+(K+)/H+逆向转运蛋白,它能够促进钠离子在液泡中积累,并且调节细胞质pH,使植株获得耐盐能力。番茄是茄科、番茄属一至两年生草本植物,栽培面积广大。番茄中含有糖类、矿物质、番茄红素等多种成分,具有健胃消食、凉血平肝、清热解毒等功效。由于番茄自花授粉、染色体图谱清楚、后代多、遗传转化操作容易、较易栽培等优点,成为生物学研究最多的模式植物之一。本研究的结果如下:(1)含有OsNHX1基因的农杆菌的对数生长期为10~15小时,选用10~15小时的农杆菌进行转化实验;(2)筛选出番茄再生培养基发芽培养基:1/2MS固体培养基;诱导培养基:MS固体培养基(3%蔗糖)+0.2mg/L IAA+2mg/L 6-BA;共培养培养基:MS固体培养基(3%蔗糖)+0.2mg/L IAA+2mg/L6-BA+0.2mg/L AS;生根培养基:MS固体培养基(3%蔗糖)+0.1mg/L NAA+100mg/L Cef(3)转化条件的确定OD600=3.0时的农杆菌重悬液,重悬液成分为MS(3%蔗糖)+0.2mg/L IAA+2mg/L 6-BA,预培养2天,侵染8分钟,共培养四天。(4)抗性植株获得及检测通过对番茄外植体进行转化,共获得抗性芽190个;生根炼苗移栽后获得抗性植株28株。经过PCR检测初步证实OsNHX1基因整合入番茄基因组中;经Southernblot杂交检测进一步证实目的基因以不同拷贝的形式整合到番茄基因组的不同位点中;经RT-PCR检测证实,抗性植株中OsNHX1基因在RNA水平表达。(5)抗性植株耐盐性检测通过对植株不同生长时期施加盐胁迫,检测抗性植株相比野生型耐盐性是否有提高,结果显示种子萌发率与野生型相比无显著差别,但平均萌发时间比野生型早2天;幼苗生长情况与野生型相比较好,不但从感受胁迫至出现枯萎的时间比野生型长,最终存活率也比野生型高。说明实验中转入的OsNHX1基因提高了番茄植株的耐盐性。本研究建立了番茄再生及转化体系,并将取自水稻的OsNHX1基因成功转入番茄中进行表达,提高了植株的耐盐性,为培育具有高耐盐性农作物打下了理论基础。但有关植物耐盐机理的研究尚未透彻,也有许多有关植物耐盐的基因还没有被克隆,因此,通过转基因的方式提高植物耐盐性仍需进行更深入的研究。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S641.2

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