不同果实特异性启动子调控下的IGF-1基因对番茄遗传转化的研究

发布时间：2018-06-17 14:16

本文选题：果实特异启动子 + 农杆菌介导法　；参考：《云南农业大学》2016年硕士论文

【摘要】：番茄(Lycopersicon esculentum Mill.)是茄科(Solanaceae),番茄属的植物,是原产自南美亚热带地区,一年生植物,是世界各地广泛种植的一种重要蔬菜作物之一。在番茄上开展表达口服疫苗的研究,可大大提升番茄的应用价值。不同类型的果实特异性启动子在果实中的发育时期及调控机理不同,筛选理想的果实特异性启动子可为利用番茄作为载体来表达外源蛋白奠定前期基础。IGF-1是调节机体生长发育、生殖、免疫和机体代谢的关键因子,在治疗糖尿病、骨质疏松、肌营养不良性侧索硬化病等疾病具有广阔的应用前景。本研究以番茄为材料,构建了包含不同果实特异性的5个启动子表达载体,采用农杆菌介导法,将IGF-1基因导入番茄中,并进行了分子检测,获得了转基因番茄植株,为利用番茄特异表达外源蛋白提供技术和理论基础,也为利用番茄口服治疗糖尿病奠定材料基础。具体研究结果如下:(1)表达载体的构建:本研究构建了5个果实特异性启动子的植物表达载体:pCAP-IGF-1、pCAPG-IGF-1、pCAGP-IGF-1、pCATP-IGF-1和pCAOC-IGF-1。并将5个植物表达载体导入农杆菌LBA4404菌株中。(2)番茄遗传转化体系的优化:试验采用AC++和1448两种番茄材料,分别进行再生体系的培养,结果表明:番茄材料AC++诱导分化的效果明显高于1448,进而确立了最佳再生体系培养的材料为番茄材料AC++。采用正交设计法,研究了不同的激素水平(IAA、ZT、AgNO3)对番茄再生体系的影响。确定了诱导愈伤组织及不定芽分化的最适宜培养基为:MS+ZT 2.0mg/L+IAA0.5mg/L+AgNO3 3.0mg/l;诱导生根的最适宜培养基为:1/2 MS+IAA 0.3mg/L。采用正交设计法,对影响转化效率的各参数(浸染液、乙酰丁香酮、浸染时间、共培养天数)进行优化,确定了最适宜的转化参数:以浸染液为MS的液体培养基,不添加乙酰丁香酮、浸染时间10 min、共培养天数2d为最佳转化条件。(3)抗性植株的PCR鉴定:经转化体系的培养,最终获得抗性苗共41株,其中AGPL1启动子获得抗性植株15株,黄瓜素启动子获得抗性植株12株,甜瓜ACO启动子获得抗性植株6株,PG启动子获得抗性植株6株,AP启动子获得2株抗性植株。对获得的抗性植株进行PCR分子检测,共21株阳性植株,阳性率达51.21%。证明外源基因IGF-1已成功整合进入番茄基因组中。其中AGPL1启动子最终获得阳性植株8株,黄瓜素启动子获得阳性植株6株,甜瓜ACO启动子获得阳性植株3株,PG启动子获得阳性植株3株,AP启动子获得1株阳性植株。
[Abstract]:Lycopersicon esculentum Mill.) Solanaceaeus, a plant of Solanaceaeus, originated from the subtropical region of South America, is an annual plant. It is one of the important vegetable crops widely planted all over the world. The study on the expression of oral vaccine on tomato can greatly enhance the application value of tomato. Different types of fruit specific promoters have different developmental stages and regulation mechanisms in the fruit. Screening ideal fruit specific promoters can lay the foundation for the expression of exogenous proteins by using tomato as a carrier. IGF-1 regulates the growth and development of the body. The key factors of reproduction, immunity and body metabolism have a broad application prospect in the treatment of diabetes mellitus, osteoporosis, muscular dystrophy, lateral sclerosis and other diseases. In this study, five promoter expression vectors containing different fruit specificity were constructed. IGF-1 gene was introduced into tomato by Agrobacterium tumefaciens, and the transgenic tomato plants were obtained by molecular detection. It provides the technical and theoretical basis for the specific expression of exogenous protein in tomato, and also lays the material foundation for oral treatment of diabetes mellitus with tomato. The specific results were as follows: in this study, five plant expression vectors of fruit specific promoters, pCAP-IGF-1, pCAPG-IGF-1, pCAGP-IGF-1pCATP-IGF-1 and pCAOC-IGF-1, were constructed. Five plant expression vectors were introduced into Agrobacterium tumefaciens LBA4404 strain. The results showed that the differentiation effect of tomato material AC was obviously higher than that of 1448, and the best regeneration system was established as tomato material AC. The effects of different hormone levels on tomato regeneration system were studied by orthogonal design. The optimum medium for callus induction and adventitious bud differentiation was: 1 MS ZT 2.0 mg / L IAA 0.5 mg / L Agno 3 3.0 mg / L, and 1 / 2 MS IAA 0.3 mg / L for rooting. Orthogonal design method was used to optimize the parameters (dipping solution, acetyl eugenone, soaking time, co-culture days) that affect the conversion efficiency. The most suitable transformation parameters were determined as follows: the liquid medium with soaking solution as MS, The resistant plants were identified by PCR without acetyl eugenone, soaking time for 10 minutes and co-culture days for 2 days. The resistant plants were identified by PCR. 41 resistant plants were obtained by culture of transformation system, 15 of which were obtained by AGPL1 promoter. 12 resistant plants were obtained by cucumber hormone promoter, 6 resistant plants were obtained by ACO promoter and 6 by PG promoter and 2 resistant plants were obtained by AP promoter. A total of 21 positive plants were detected by PCR, and the positive rate was 51.21%. It was proved that the exogenous gene IGF-1 had been successfully integrated into tomato genome. Among them, 8 positive plants were obtained from AGPL1 promoter, 6 positive plants from cucumber hormone promoter, 3 positive plants from ACO promoter of muskmelon, 3 positive plants from PG promoter and 1 positive plant from AP promoter.
【学位授予单位】：云南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S641.2

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