【作者】 孟颢光；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2011， 博士

【摘要】 梨是世界上最重要的温带水果之一,属于蔷薇科,广泛种植于温带和亚热带地区。在梨树传统的杂交育种中存在许多问题,这些问题包括童期长、杂合程度高和在育种选育中需要种植的面积大,这些不利条件严重阻碍了其育种进程。近年来,随着植物基因工程的不断发展,特别是农杆菌介导的遗传转化方法在作物育种上的广泛应用,就可以将外源的具有经济价值的基因整合进目标作物,为改良其某一性状提供资源。农杆菌介导的遗传转化主要依赖于受体材料的再生能力及农杆菌的转化效率,因此,建立良好而高效的再生体系是遗传转化成功的关键。本试验以砀山酥梨离体叶片为材料,研究基本培养基类型、激素种类和浓度配比、接种方式、苗龄、暗培养时间和硝酸银对砀山酥梨离体叶片再生不定芽的影响,优化了砀山酥梨离体叶片的再生体系,并在此基础上建立农杆菌介导的遗传转化体系。通过农杆菌介导法将来源于华东野生葡萄(Vitis pseudoreticulata W. T. Wang)的株系“白河-35-1”的VpSTS和VpPR10基因导入砀山酥梨的基因组中。取得了以下主要结果:1.建立了砀山酥梨离体叶片再生体系比较了6种基本培养基,MS、1/2MS、QL、NN69、WPM和B5,NN69基本培养基适合砀山酥梨离体叶片的再生。BA(3.0 mg/L)和IBA(0.2 mg/L)组合对不定芽的再生有很好的促进作用。一定时间的暗培养也能促进砀山酥梨不定芽的再生,一般以3周的暗培养处理效果最佳;不同的叶龄的叶片不定芽再生率有显著的差异,25 d叶龄的叶片得到的再生率最高;叶片远轴面向下接触培养基;再生培养基中附加0.5 mg/L的AgNO3对砀山酥梨的不定芽再生都有显著地影响。2.构建了植物表达载体pWR-II-VpPR10将质粒pCAMBIA1300和pWR306经限制性内切酶HindⅢ/EcoRⅠ双酶切后,再定向连接,得到含有ED35s启动子、Omega元件、MCS及TNOS终止子的中间表达载体pWR-Ⅱ;再将质粒pWR-Ⅱ和pGEM-Teasy-VpPR10经限制性内切酶BamHI/SalI双酶切后,再定向连接,得到以潮霉素作为筛选标记基因的植物表达载体pWR-II-VpPR10,并用改进的冻融法导入根癌农杆菌EHA105。3.建立了砀山酥梨的遗传转化体系在砀山酥梨的转化体系中,潮霉素的筛选临界浓度为5.0 mg/L,头孢霉素抑菌浓度为300 mg/L。试验中研究了一些影响转化的因素:菌液浓度和侵染时间、共培养时间、AS浓度等等。得到了转化最优方案:菌液浓度OD600为0.5,侵染5 min,共培养48 h,AS浓度100μM,延迟5 d的筛选方式。采用两步生根法诱导转基因植株生根,试管苗在附加80 mg/L PG、20 mg/L IBA的ASH基本培养基上暗培养7 d后转入附加80 mg/L PG的ASH培养基上进行光照培养,生根率达到91.7%。4.转基因植株检测潮霉素的抗性植株经过PCR检测、Southern blot杂交检测和RT-PCR检测,证明VpSTS和VpPR10基因已整合到砀山酥梨基因组中,对转VpSTS基因呈阳性的植株进行进一步的高效液相色谱(HPLC)检测显示,其中的3个转基因株系中白藜芦醇的含量分别为1.50μg/g(鲜重)、2.137μg/g(鲜重)、1.931μg/g(鲜重),而非转化植株中没有检测到白藜芦醇。更多还原

【Abstract】 Pear is one of the most important temperate fruit crops. It belongs to the Rosaceae family, and is widely grown in temperate and sub-tropical regions of the world. There are many problems in the traditional cross-breeding of pear tree, including a long juvenile period, high levels of genetic heterozygosity, and the need for a large area to screen ideal cultivars. These seriously hamper the process of pear breeding. With the development of plant genetic engineering, Agrobacterium-mediated genetic transformation method was widely used in crop breeding. It can make some economic value exogenous gene(s) integrate into the crop, to improve desirable traits for target plants. An efficient regeneration system is thought to be crucial to the success of plant genetic engineering, since the efficiency of Agrobacterium-mediated transformation is considered to be dependent on two primary factors, one being the regeneration ability of the infected tissue, and the other, the infection efficiency of Agrobacterium.In this research, we have studied the effects of a series of factors (basic media, plant growth regulators, explants inoculation methods, leaf ages dark period and AgNO3) in the inducing adventitious shoot of Dangshansu pear. Then on the basic of optional regeneration system obtained above, we established an efficient and quick Agrobacterium-mediated transgenic system of Dangshansu pear, with which the VpSTS and VpPR10 gene from Chinese wild Vitis pseudoreticulata accession Baihe-35-1 were respectively transformed into the Dangshansu pear genome. The details were as follows:1. Establishment of the regeneration system from leaves of Dangshansu pear in vitro Compared with MS, 1/2MS, QL, NN69, WPM and B5 medium, NN69 medium was more suitable for Dangshansu pear leaf regeneration.The combination of BA 3.0 mg/L and IBA 0.2 mg/L has a good role in promoting shoot regeneration.A certain period of dark culture can also promote the regeneration of Dangshansu pear adventitious buds, among which 3-week dark culture treatment was the best. There was a significant difference in the leaf shoot regeneration rates with various leaf ages. Among them, the leaves with 25-d ages displayed the highest regeneration rate. It was also found that leaf abaxial face down in contact with the medium and regeneration medium supplemented with 0.5 mg/L of AgNO3 have significant impact on shoot regeneration Dangshansu pear. 2. Construction of the plant expression vector pWR-II-VpPR10A nucleotide sequence carrying promotor ED35s, Omega TMV translation enhancer and nopaline synthase terminator was cut from plasmid pWR306 and specifically cloned intoHindⅢ/EcoRⅠdigested plasmid pCAMBIA1300 to construct intermediate vector pWR-II. The segment of VpPR10 gene from the recombinant plasmid pGEM-T-VpPR10 was specifically cloned into BamHI/ SalI digested pWR-II to construct the plant expression vector pWR-II-VpPR10. The recombinant plant expression vector were introduced into Agrobacterium strain EHA105 using the improved freezing-thawing method.3. Establishment of an efficient Dangshansu pear transformation systemIn the transformation of Dangshansu pear, the critical concentration of hygromycin selection was 5 0 mg/L and the inhibitory concentration of cefotaxime was 300 mg/L. Some factors that affect the transformation of Dangshansu pear were studied, including bacterium concentration, infecting time, co-culture time and AS concentration. An optimal transformation protocol was obtained and described as followings: OD600=0.5, 5 min for infection, 48 h for co-culture, 100μM AS in co-culture medium, and 5-d delayed selection. The best induction root was achieved by two-step rooting method. First, the plantlets in vitro were incubated on ASH medium supplemented with 80 mg/L PG and 20 mg/L IBA and cultured in the dark for 7 days. Second, the shoots were transferred to the same medium with 80 mg/L PG in the normal light conditions. As a result, 91.7% of the rooting rate was obtained under above conditions.4. Molecular detection of transgenic plantsHygromycin-resistant transgenic pear plants were validated using polymerase chain reaction (PCR), RT-PCR and Southern blot analysis. The result indicated that the VpSTS and VpPR10 gene have been integrated into the Dangshansu pear genome. The positive plants transformed with VpSTS gene were further analyzed using high performance liquid chromatography (HPLC) method. The result displayed that the resveratrol contents of 3 tested transgenic pear plants were 1.50μg/g, 2.137μg/g, 1.931μg/g (fresh weight), whereas non-transgenic plants were not detected in resveratrol.更多还原