新疆野苹果再生体系、遗传转化体系的建立及CRISPR/Cas9 编辑系统的初探
Alternative TitleEstablishment of in vitro regeneration, genetic transformation and CRISPR/Cas9 gene editing systems in Malus Sieversii
Thesis Advisor张道远
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline工程硕士
Keyword新疆野苹果 再生体系 遗传转化体系 CRISPR/Cas9基因编辑 Xinjiang wild apple Regeneration Transformation CRISPR/Cas9
Abstract新疆野苹果(Malus sieversii)又名塞威士苹果, 隶属蔷薇科苹果属, 是世界栽培苹果的祖先种。新疆野苹果是第三纪孑遗物种,因其自交不亲和特性和自然选择的作用,遗传资源丰富。 以新疆野苹果为主要建群种的天山野果林是我国重要的自然林,在调节气候、涵养水源、保持水土等方面有着重要的生态作用。 近些年来由于农田开发、过度放牧、人为砍伐、苹小吉丁虫危害以及腐烂病爆发等原因,使得新疆野苹果群落面积急剧减少, 本物种已被列为国家濒危二级保护植物,有效保护这一宝贵种质迫在眉睫。为保存优质种质及其基因资源开发利用, 本研究以新疆野苹果为材料,探究了其再生体系、遗传转化体系及 CRISPR/Cas9 基因编辑系统在本物种中的应用。 结果如下:1. 建立了完整的基于愈伤组织分化的新疆野苹果再生体系。以新疆野苹果幼嫩叶片和非茎尖茎段为外植体,经过愈伤组织诱导及不定芽的再生、芽增殖、生根、壮苗和移栽过程,首次建立了完整的基于愈伤组织分化的新疆野苹果的再生体系,最短成苗时间为 2.5 个月。其中, 新疆野苹果叶片最佳诱导再生培养基为 MS+4mg/LTDZ+1mg/L NAA,茎段最佳诱导再生培养基为 MS+1mg/L 6-BA, 叶片和茎段不定芽最高再生率分别为 35%、 90%。 继代培养基为 MS+0.4mg/L 6-BA+0.1mg/L NAA,最佳生根培养基为 1/2MS+0.1mg/L NAA+15g/L sucrose。2. 建立了根癌农杆菌介导的新疆野苹果叶盘转化法。确定了 Kan 和 Hyg 两种抗生素的筛选压分别为 15mg/L, 4mg/L。确定了一种抑菌剂 Cef 的工作浓度(400mg/L);对比了不同的浸染液、浸染时间以及叶片和农杆菌共同培养方式,确定了农杆菌浸染液为: MS+3% sucrose+150μM AS,共培养及延迟筛选时间分别为 2d为较好的培养方式。通过这种方法, 已获得了大量转基因的愈伤组织。筛选压的存在,会强烈抑制愈伤组织和不定芽的再生,所以又进一步探究了前期不添加筛选压进行稳定转化的可行性: 叶片外植体受到农杆菌浸染后培养基中不添加筛选压,待不定芽长出后再对不定芽进行筛选,已获得了阳性幼芽。3. 建立了新疆野苹果的 CRISPR/Cas9 基因编辑体系。本实验以 MsPDS 为靶标基因,此基因功能缺失会造成植物白化表型,设计了 5 个靶向不同位点的 sgRNA,共构建了 pYL/Cas9, pPTG/Cas9 两类共 8 种不同的载体,转化了叶片外植体,获得了一些阳性的愈伤组织和不定芽。获得了一些白化表型的愈伤组织,并通过测序检测到此基因被编辑的位点附近出现了不同程度的碱基的缺失,证明 CRISPR/Cas9 基因编辑系统可以在新疆野苹果物种中工作。
Other AbstractXinjiang wild apple (Malus sieversii), belonging to apple genus, is the origin speciesof cultivated apple in the world. Xinjiang wild apple is a relict species. Because of itsself-incompatibility and the role of natural selection, Xinjiang wild apple is rich in geneticresources. Tianshan Wild Fruit Forest, an important natural forest in China, plays animportant ecological role in regulating climate, conserving water resources andpreserving soil and water. In recent years, however Xinjiang wild apple populationquantity decrease dramatically due to serious disease and insect pests infestation and hasbeen listed as a national endangered plant. It is urgent to protect this precious germplasmeffectively.It is of great significance for the conservation of Xinjiang wild apple and theexploitation and utilization of its gene resources. So in this study, the regeneration system,genetic transformation system and CRISPR/Cas9 editing system of Xinjiang wild applewere studied. The results showed as follows:1. A complete regeneration system based on callus differentiation was established.Young leaves and stem segments of Xinjiang wild apple were used as explants. Theprocess include callus induction and adventitious bud regeneration, prolongation, rooting,acclimatization and transplanting. It will take 2.5 months to complete the process. Theoptimum medium for leaf regeneration was MS+4mg/L TDZ+1mg/L NAA, the bestmedium for stem regeneration was MS+1mg/L 6-BA, the shoot propagation medium forelongation was MS+0.4mg/L 6-BA+0.1mg/L NAA. The best rooting medium was1/2MS+0.1mg/L NAA+15g/L sucrose. The highest regeneration rate of adventitious budsin leaves and stem segments was 35% and 90%, respectively.2. The agrobacterium-mediated genetic transformation system of Xinjiang wildapple was established using leaf disk. The selective pressures of two antibiotics, Kan andHyg, were determined to be 15 mg/L and 4 mg/L, respectively. The concentration Cef, akind of bacteriostatic agent, is 400 mg/L. The components of the Agrobacteriumsuspensions include MS+3% sucrose+150 μM AS. The optimum transformationconditions were explants and Agrobacterium firstly co-cultured for 2 days and delayedselection for another 2 days. Until now, a large number of transgenic calli have been obtained. It is time-consuming to obtain buds from callus because of the existence ofantibiotic. On this basis, the feasibility of another transformation without addingscreening pressure is explored. The explants were firstly transformed, growing in themedium without antibiotic. After the buds appearing, they were transferred into themedium with an antibiotic. Based on this idea, I got a small amount of positiveadventitious bud on the medium with antibiotic.3. The CRISPIR/Cas9 gene editing system was applied to Xinjiang wild apple forthe first time. In this experiment, MsPDS was used as the target gene, and the deletionfunction of this gene could cause the albinism phenotype. Five sgRNAs targeted atdifferent extron were designed, and both were further constructed into differentpaired-sgRNA/Cas9 binary vector. The leaf blade was transformed by theabove-mentioned 2 method. Some positive calli and adventitious buds were obtained.There were a little callus and adventitious buds with albinism phenotype. Some basesmissed or inserted around the target loci by sanger sequencing. The CRISPR/Cas9 geneediting system in Xinjiang wild apple was established successfully.
Subject Area生物工程
Document Type学位论文
First Author Affilication中国科学院新疆生态与地理研究所
Recommended Citation
GB/T 7714
张燕. 新疆野苹果再生体系、遗传转化体系的建立及CRISPR/Cas9 编辑系统的初探[D]. 北京. 中国科学院大学,2019.
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