【作者】 王佳；

【导师】 王飞；

【作者基本信息】 西北农林科技大学 ， 果树学， 2009， 硕士

【摘要】 我国是猕猴桃的起源和分布中心,资源极其丰富。猕猴桃果实风味独特,营养价值高,同时具有医疗效果,深受人们喜爱。随着生活水平和消费结构的变化,人们对猕猴桃的需求量越来越大,但是现有猕猴桃栽培品种果实难以保鲜、不耐贮运一直是猕猴桃采收、运输、销售过程中的一个难题。因此,培育耐贮运的猕猴桃新品种,解决生产与消费上的实际问题,成为科学家们研究的主要课题之一。由于常规育种的局限性、时间性以及化学保鲜剂的毒副作用,近年来,生物技术的发展,为克服传统育种的缺陷和化学保鲜剂使用的毒副作用提供了新的育种观念、方法和手段。本研究是在构建好的ACO氧化酶基因干扰载体的基础上,以中华猕猴桃“西选二号”为试验材料,建立起叶片和茎段的再生体系以及遗传转化体系,利用根癌农杆菌介导法将从百合中克隆到的ACC氧化酶基因片段采用RNA干扰技术导入猕猴桃外植体,通过PCR检测获得了阳性表达植株,为后续试验提供了宝贵试验材料。主要研究结果如下:1.建立了猕猴桃“西选二号”叶片和茎段的再生体系以猕猴桃“西选二号”田间取材的叶片和茎段为外植体,通过不同植物激素种类组合和浓度配比诱导产生愈伤组织并且分化不定芽。结果表明,叶片和茎段在下面培养基中均能诱导出愈伤组织,但是诱导效果不同。(1)叶片诱导愈伤组织的适宜培养基为MS+0.5mg.L-16-BA+0.25mg.L-1NAA,诱导频率为89.1%;以MS+0.5mg.L-16-BA+0.1mg.L-1NAA诱导茎段形成愈伤组织效果较好,诱导频率达93%;(2)愈伤组织分化不定芽的最适培养基为MS+3.0mg.L-16-BA +0.1mg.L-1NAA,出芽频率达92%;继代增殖培养基为MS+3.0mg.L-16-BA+ 0.1mg.L-1NAA+ GA30.6mg.L-1;(3)生根培养基为1/2MS+1.0mg.L-1IBA+0.2 mg.L-1NAA,根长势粗壮,移栽成活率高。2.遗传转化受体系统的建立以猕猴桃“西选二号”再生形成的叶片为外植体,建立了遗传转化系统。(1)叶片诱导愈伤组织的最适培养基为MS+0.5mg.L-16-BA+0.25mg.L-1NAA,愈伤组织分化不定芽的最适培养基为MS+3.0mg.L-16-BA +0.1mg.L-1NAA;(2)卡那霉素筛选压力的确定:叶片作为转基因的受体材料时,卡那霉素的致死浓度为20 mg.L-1;(3)抑菌素种类和浓度的确定:头孢霉素霉素浓度为300 mg.L-1时能有效抑制农杆菌的生长并不影响外植体的分化。3.遗传转化利用根癌农杆菌介导法对猕猴桃转化过程中,对预培养时间、农杆菌菌液浓度等影响因子进行了研究。叶片经6d预培养,于OD600值为0.6的菌液中侵染8min,在含有20mg.L-1的乙酰丁香酮的培养基上共培养3d,转移到选择培养基中进行培养。4.转基因植株的获得及检测研究共获得卡那霉素抗性植株68株。对其进行PCR检测,有5株扩增出目标条带,初步证明外源基因已经部分整合到这5株基因组中。更多还原

【Abstract】 China was the original and distributed resource centre of Actinidia. Kiwifruit had a niceTaste、high nutrition value and medical effects, kiwifruit attracts more attention in present years.Along with the improvement of living standard and changes of consumption structure, the requirement to high quality kiwifruit is increasing year after year. One problem in kiwifruit production is liable to a post-harvest deterioration that impairs their preservation and is cause of great losses.In this case,The research area of scientists is breeding long keeping variety by transferring foreign gene.With the limit of common hybridization and negative effection of chemical on fruits.Biotechlonogy development provided new concepts,new methods and new tools to overcome the shortcomings of traditional breeding and the use of the preservative chemical toxicity.On the basis of the ACC Oxidase RNAi gene expression vectors built, The Acitinidia Xixuan No.2 use as explants, the experiment optimized tissue culture system of kiwifruit;carried on systematic research to kiwifruit Agrobacterium tumefaciens-mediated genetic transformation.The foreign gene ACO which cloned from lily was built as dsRNA-mediated gene silencing vector and transformed by Agrobacterium EHA105.The purpose of this study is to achieve transgenic lily of ACC Oxidase RNAi gene and provide some beneficial foundation in kiwifruit in the future.The main results were as follows:1.A regeneration sysem of leaf and nod was initiated from Acitinidia Xixuan No.2Leaves and nods of Acitinidia Xixuan No.2 were chosen as explants.Different type and concentration of hormone were studied in order to induce callus and adventitious.The results showed that,The leaf and nod could grow callus in all treatment,but induced results were different.(1)The optimum medium for inducing callus from leaves was MS+0.5mg.L-16-BA+0.25 mg.L-1NAA,the inducing frequency may reach 89.1%; The combination of MS+0.5mg.L- 16-BA+0.1mg.L-1NAA was the appropriate medium for nods forming callus with the inducing rate 93%.(2)The best medium for the buds induction was MS supplement with 3.0mg.L-16-BA and 0.1mg.L-1NAA;The best combinations of shoot proliferation was MS+3.0mg.L-16-BA +0.1mg.L-1NAA+ 0.6 mg.L-1GA3;(3)The medium of 1/2MS +1.0mg.L-1IBA + 0.2mg.L-1NAA for rooting with strong roots.2. Establishment of High-efficiency acceptor system for the gene transformation Acitinidia Xixuan No.2Taken the regeneration leaf as explants,establish the acceptor system.(1)The combination of MS+0.5mg.L-16-BA+0.25mg.L-1NAA was the optimum medium for the induction of callus;the medium of MS+3.0mg.L-16-BA +0.1mg.L-1NAA was the best medium for regeneration buds from callus(2) The concentrations of kanamycin were 20 mg.L-1.(3) The Cefotaxime Sodium used for eliminating Agrobacterium and the optimum concentration was 300 mg.L-1.3. Genetic transformationwe studied the factors that affected transformation ratio of Agrobacterium-mediated method.Leaf explants were pre-culture for 6 days、8 min infection, 0.6 OD600;3 days co-culture.The co-cultivation was carried out on medium containing 20mg.L-1 acetosying one(AS).4 Identification of transdenic plantsIn this study,68 kanamycin resistant were obtained. Selected for PCR,5 plantlets got the strip.It proved that foreign gene had been integrated into these 5 plants genome.更多还原