抗寒基因的克隆及根癌农杆菌介导的ICE1基因转化柑橘的研究

Cold Tolerance Gene Cloning and Agrobacterium-mediated ICE1 Gene Transfer in Citrus

作者: 专业:果树学 导师:孙中海 年度:2005 学位:博士 

关键词
上胚轴 成年态茎段 抗寒 农杆菌介导的转化 ICEI基因

Keywords
Epicotyl segments, mature stem segments, cold tolerance, Agrobacterium-mediated transformation, ICE1 gene
        柑橘是世界上最重要的果树之一,在我国水果产业中占据了重要的经济地位。然而,周期性的冻害严重影响了柑橘的产量和品质,这个问题的解决有赖于柑橘的抗寒育种和抗寒栽培。柑橘类植物的多胚性、遗传背景复杂、童期长、单性结实的特点限制了柑橘常规育种的发展。柑橘组织培养以及建立在此基础上的遗传转化为缩短育种周期、获得抗寒、优质柑橘种质资源提供了新的途径。 本实验以冷敏感的尤力克柠檬(Citrus limon(L.)Burm.f.cv.Eureka)上胚轴为材料,建立了高效的再生体系;以田间茎段为材料,建立了我国大力推广的栽培品种—纽荷尔脐橙(C.sinensis(L.)Osbeck cv.Newhall)的成年态组织培养体系,为成年态柑橘的遗传转化打下了基础。利用从拟南芥中克隆的ICE1基因构建了植物表达载体,并通过根癌农杆菌介导的方法将该基因导入了尤力克柠檬中,初步建立了根癌农杆菌介导的柠檬实生苗转化体系。 主要研究结果如下: 1)研究了影响柠檬(C.limon(L.)Burm.f.cv.Eureka)不定芽再生的主要因素。结果表明:外植体类型,培养基中生长调节剂浓度配比,暗培养时间对柠檬不定芽的再生有显著的影响。上胚轴是再生不定芽的最佳外植体类型。上胚轴在基本培养基中能形成不定芽,BAP和NAA的加入则有利于不定芽的再生。当BAP和NAA浓度分别为1.0和0.1 mg L-1时,上胚轴不定芽分化率达到76.6%,此时不定芽数也达到最高,为9.25个。暗培养20 d有利于不定芽数的提高,最高达到每个上胚轴13.46个。不定梢在附加IBA或NAA的MT培养基中均可生成不定根。IBA有利于不定根的伸长,NAA有利于不定根数量的增加。 2)利用纽荷尔脐橙(C.sinensis(L.)Osbeck cv.Newhall)田间成年态茎段为材料,建立了高效的植株再生体系。实验结果表明:纽荷尔脐橙节段以及节间茎段均可通过器官发生途径再生不定芽。光照条件和植物生长调节剂的组合影响了不定芽的再生,在1.0 mg L-1 BAP和0.5 mg L-1 NAA的培养基中,15 d暗培养有利于不定芽的再生,此时节间茎段不定芽的再生率达到了85.2%,平均每个外植体上产生了3.7个不定芽。伸长的不定芽在附加0.5或1.0 mg L-1NAA的1/2MT生根培养基中生根并移栽成活。 3)根据GeneBank中已验证功能的甘油酸酰基转移酶(GPAT)基因序列以及类GPAT基因的Expression sequence tag(EST)设计一对兼并引物,扩增出了一条长约500 bp的片段,生物信息学分析表明该片段和已知的GPAT基因序列以及类GPAT基因没有明显的相似性,说明扩增的序列非目的片段。在文中对出现这种情
    Citrus is one of the most important fruit trees in the world, and it also holds an important position in the fruit tree industry in China, especially in the southern China. However, periodic freezing injury seriously affected the yield and quality of citrus fruits. It depends on the breeding program to develop cold tolerant citrus cultivars to solve this problem. Conventional breeding plays a limited role in this program because citrus has characteristics such as poly embryo, complex inheritance background as well as male or female sterility. Tissue culture and transgenic research shortened the breeding program cycle, thus provided a promising way to obtain elite citrus germplasm that are cold tolerant.In present research work, various factors that affected the adventitious buds regeneration from cold sensitive "Eureka" lemon (C. limon (L.) Burm. f. cv. Eureka) were investigated. Tissue culture system using mature stem segments derived from Newhall navel orange (C. sinensis (L.) Osbeck cv. Newhall) was established, which provides a platform for transgenic study using explants derived from mature citrus. Plant expression vector was constructed using ICE1 that was cloned from Arabidopsis, and this gene was successfully transferred into the lemon genome mediated by Agrobacterium, and the preliminary optimized transformation protocol for lemon epicotyls was developed.The main results of our research work are as follows:1) Main factors that affected the lemon (C. limon (L.) Burm. f. cv. Eureka) adventitious buds regeneration were investigated. The results showed that some factors such as explant types, the combination of the kinds and concentration of plant growth regulators in the culture media and the time of dark treatment, significantly affected lemon adventitious buds regeneration. Epicotyl segments were the optimal explants for adventitious buds regeneration. Adventitious buds were regenerated from epicotyl segments in MT basal medium, and the addition of BAP and NAA in the media influenced the buds regeneration. When BAP and NAA added to the medium at 1.0 and 0.1 mg L~-1 respectively, regeneration frequency of epicotyl segments reached 76.6%, at the same time, 9.25 buds per explant were obtained. 20 d darkness incubation enhanced the adventitious bud regeneration, which produced 13.46 adventitious buds per epicotylsegment under optimal plant growth regulator agenda. With the application of either IBA or NAA, the adventitious shoots developed roots, with IBA elongated the root length and NAA increased the root number.2) An efficient in vitro regeneration system through direct organogenesis from mature nodal and intemodal stem segments of Newhall navel orange (C. sinensis (L.) Osbeck cv. Newhall) was established. Illuminating conditions together with plant growth regulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation increased the adventitious bud regeneration, and the highest regeneration frequency (85.2%) and bud formation efficiency (3.7 per responsive internodal stem segment) were obtained when 1.0 mg L~-1 BAP and 0.5 mg L~-1 NAA was supplemented. The elongated adventitious buds were successfully rooted in 1/2 MT media supplemented with 1.0 mg L~-1 NAA and transferred to the soil.3) Based on the GPAT gene sequences deposited in Genebank or their analogues ESTs, a pair of degenerate primers were designed, and subsequently a fragment about 500 bp was amplified using Poncirus genome as template. Bioinformatic analysis indicated that the cloned fragment was not the target sequence for its sequence similarity with other GPAT genes is pretty low. Possible reasons were discussed and preliminary methods to solve the problem were proposed. A pair of primers was designed for the Arabidopsis ICEl gene, and subsequent RT-PCR amplified the fragment of ICEl gene with its coding sequence region. Based on the plant expression vector pMV, another vector nominated pMVICEl was constructed, in which ICEl gene was regulated by CaMV 35S promoter and NOS terminator. This binary vector was successfully transferred to Agrobacterium tumafaciency strain EHA105.4) Factors that affected the transformation frequency of lemon epicotyl were investigated. The results showed that younger explants are more readily to be transformed than older ones. The time (7.5-30 min) that explant immersed in the Agrobacterium suspension cells did not significantly affected the transformation frequency. 100 uM or 200 uM acetosysingone in the coculture media enhanced the transformation frequency when lemon epicotyls were used as explants. Epicotyl segments precultured in darkness for a short period of time enhanced transformation frequency, however, prolonged dark treatment inhibited it. The results also demonstrated that 19℃ coculture temperature, 5 d coculture in the media as well as tobacco feeder plate and the addition of 2,4-D in coculture media improved the lemon epicotyltransformation frequency. The highest transformation frequency amounted to 15.4% in our experiment, which was greatly enhanced compared with previous reports that used lemon stem segment as explants. PCR and Southern blot analysis indicated that ICE1 gene was successfully integrated into lemon genome, and the target gene was inserted into more than one place in lemon genome. Finally, 7 transgenic lemon seedlings were obtained and 5 of them were successfully transferred to soil.
        

抗寒基因的克隆及根癌农杆菌介导的ICE1基因转化柑橘的研究

中文摘要9-11
Abstract11
第一章 综述14-35
    第一节 柑橘转基因研究进展14-26
        1 植物转基因历史和现状14
        2 转基因方法14-20
            2.1 DNA直接导入法15-16
                2.1.1 化学诱导的DNA直接转化法15-16
                2.1.2 物理诱导的DNA直接转化法16
            2.2 载体介导的DNA导入法16-20
                2.2.1 根癌农杆菌的生物学特性16-17
                2.2.2 根癌农杆菌 Ti质粒的结构和功能17
                2.2.3 根癌农杆菌介导的T- DNA在植物中的转化17-18
                2.2.4 农杆菌 T- DNA质粒载体系统18
                2.2.5 影响农杆菌转化效率的因素18-20
                    2.2.5.1 农杆菌菌株的选择18-19
                    2.2.5.2 诱导条件的选择19-20
                    2.2.5.3 外植体的选择和预处理20
                    2.2.5.4 抗生素的选择20
        3 柑橘遗传转化发展现状及存在的问题20-26
            3.1 柑橘遗传转化发展现状20-24
            3.2 柑橘遗传转化存在的问题24-26
                3.2.1 转化中使用的外植体类型24
                3.2.2 抗性芽的生根24-25
                3.2.3 转化使用的目的基因25-26
    第二节 植物抗寒基因工程26-33
        1 抗冻蛋白26-27
        2 小分子渗透调节剂27-28
        3 脂肪酸去饱和酶28
        4 抗氧化酶28-29
        5 抗寒锻炼相关基因29-32
        6 柑橘抗寒育种研究进展和局限性32-33
    第三节 课题的提出33-35
        1 柑橘抗寒基因工程育种研究的必要性33-34
        2 本研究的目的意义34-35
第二章 尤力克柠檬组织培养体系的建立35-42
    1 前言35-36
    2 材料和方法36
        2.1 不同外植体不定芽再生的诱导36
        2.2 上胚轴不定芽诱导条件的优化36
        2.3 不定芽生根36
        2.4 培养条件36
    3 结果36-40
        3.1 不同外植体的不定芽再生率36-37
        3.2 植物生长调节剂对不定芽再生的影响37-38
        3.3 暗培养对柠檬上胚轴切段不定芽再生的影响38-39
        3.4 不定芽生根39-40
    4 讨论40-42
第三章 纽荷尔脐橙成年茎段器官直接发生途径再生不定芽42-48
    1 前言42
    2 材料和方法42-43
        2.1 材料42
        2.2 培养条件42-43
        2.3 实验设置和数据处理43
    3 结果43-46
        3.1 外植体、光照以及 BAP和 NAA对不定芽再生影响43-45
        3.2 不定芽生根45-46
    4 讨论46-48
第四章 植物抗寒基因的克隆以及植物表达载体的构建48-66
    1 前言48-49
    2 材料和方法49-51
        2.1 实验材料49
            2.1.1 植物材料49
            2.1.2 菌株、质粒以及实验药品49
        2.2 方法49-51
            2.2.1 积壳叶片总 DNA的提取49
            2.2.2 设计兼并引物扩增枳壳 GPAT基因片段49
            2.2.3 扩增GPAT基因片段的条件49
            2.2.4 总RNA的提取49
            2.2.5 cDNA的合成49-50
            2.2.6 PCR扩增ICE1编码区50
            2.2.7 扩增产物的克隆以及序列分析50
            2.2.8 质粒提取50
            2.2.9 ICE1基因表达载体的构建50
            2.2.10 pMVICE1转化农杆菌50-51
    3 结果51-64
        3.1 枳壳 GPAT基因的引物设计51-52
        3.2 枳壳 GPAT基因片段的扩增和序列分析52-53
        3.3 拟南芥 RNA的提取与 RT-PCR扩增ICE1基因片段53-54
        3.4 ICE1基因片段序列测定和分析54-63
        3.5 ICE1基因的酶切及表达载体的构建63-64
    4 讨论64-66
第五章 根癌农杆菌介导的柠檬上胚轴转化 ICE1基因研究66-79
    1 前言66
    2 材料和方法66-69
        2.1 实验材料66-67
            2.1.1 供试植物材料66
            2.1.2 农杆菌和质粒66-67
            2.1.3 主要生化试剂67
        2.2 培养条件及不同的培养基配方67
        2.3 实验方法67-69
            2.3.1 柠檬上胚轴对卡那霉素、头孢霉素敏感性67
            2.3.2 菌液的准备67-68
            2.3.3 柠檬上胚轴遗传转化过程68
            2.3.4 遗传转化条件的选择68
            2.3.5 抗性芽的生根与移栽68
            2.3.6 PCR鉴定68-69
            2.3.7 转化效率的数据统计69
            2.3.8 Southern杂交验证69
    3 结果69-74
        3.1 抗生素对柠檬上胚轴不定芽再生的影响69-70
        3.2 影响柠檬上胚轴转化效率的因素分析70-73
            3.2.1 外植体苗龄70
            3.2.2 外植体预培养时间70-71
            3.2.3 外植体在农杆菌中的侵染时间71
            3.2.4 乙酰丁香酮浓度71-72
            3.2.5 共培养时间72
            3.2.6 共培养温度72-73
            3.2.7 共培养培养基73
        3.3 柠檬上胚轴的转化及植株再生73-74
        3.4 转化植株的分子检测74
    4 讨论74-79
第六章 问题和展望79-83
    1 柑橘理想的转化系统79
    2 柑橘转基因育种79-80
    3 柑橘转化中使用外植体80
    4 转化中目的基因的讨论80-81
    5 柑橘抗寒基因工程81-82
    6 转基因安全性的讨论82-83
参考文献83-94
附录Ⅰ 常用分子生物学操作94-99
附录Ⅱ 博士在读期间发表的文章99-100
致谢100


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