【作者】 曾晓芳；

【导师】 赵德刚；

【作者基本信息】 贵州大学 ， 植物学， 2008， 硕士

【摘要】 本研究以朝仓花椒为研究对象,建立组培再生体系,并在此基础上,首次利用农杆菌介导法将细胞分裂素生物合成限速酶—异戊烯基转移酶基因（ipt）导入花椒体内,探索花椒遗传转化的技术参数,初步建立了花椒遗传转化体系,为利用基因工程技术对花椒进行遗传改良打下基础。其主要研究内容如下:1花椒再生体系建立试验以花椒萌发生长5-20d的无菌苗叶柄、茎段及叶片为外植体,研究在以WPM培养基为基本培养基中附加不同的浓度的细胞分裂素（6-BA）和生长素（NAA、IBA）,对朝仓花椒愈伤组织诱导及再生的影响,建立了朝仓花椒植株再生体系。结果表明:愈伤组织诱导及分化最适培养基为WPM+6-BA0.5 mg·L^-1+NAA0.2 mg·L^-1,最适生根培养基为1/2WPM不添加任何激素,移栽时,以珍珠岩和田土1∶3比例成活率最高,28d后统计成活率可达85.6%。2农杆菌介导的朝仓花椒遗传转化体系建立建立了以朝仓花椒叶柄及茎段为转化受体的根癌农杆菌遗传转化体系,初步确定了抗生素卡那霉素（kan）的筛选浓度,在遗传转化过程中,详细探讨了外植体预培养时间、浸染菌液浓度、浸染时间、共培养时间等影响因素对转化效率的影响。结果表明:外植体预培养3d,菌液浓度在OD值0.5-0.7,浸染10min,共培养3d为最佳技术参数;延迟7d筛选可提高转化效率,重悬液及共培养基中添加100μmol·L^-1AS可提高转化率至24.6%。3转化植株检测通过农杆菌介导法遗传转化朝仓花椒,并对转化植株进行GUS组织活性及PCR检测。结果表明部分转化植株GUS染色显蓝色,部分植株PCR检测呈阳性,初步证实目的基因已转入花椒基因组中。ipt基因表达有效促进了朝仓花椒遗传转化过程中不定芽的诱导,转ipt基因花椒能产生大量分枝,易于增殖培养,但转基因植株生根受到抑制。更多还原

【Abstract】 In this study,an efficient plant regeneration protocol in vitro and transformation with ipt gene mediated via Agrobacterium tumefaciens method of Zanthoxylum piperitum DC var.inerme Makino were achieved firstly.The establishment of efficient regeneration system and Agrobacterium tumefaciens-mediat -ed transformation of Zanthoxylum piperitum DC var.inerme Makino will make substantial contribution as an essential and useful means to further fundamental study of breeding by the application of genetic engineering technology.1.Establishment of high efficient regeneration system of Zanthoxylum piperitum DC var. inerme MakinoIn this study,the explants（leave petioles and stems） were cultured in vitro on WPM medium supplanted with N-6benzyladenine（6-BA）,α-naphthaleneacetic acid（NAA） and indole-3-butyric acid （IBA）,and established the plant regeneration system of Zanthoxylum piperitum DC vat.inerme Makino. The results showed that the most suitable culture medium for callus induction and shoots reproduction was WPM+6-BA0.5 mg·L^-1+NAA0.2 mg·L^-1.Induction of rooting was achieved by transferring the shoots to the half strength macro-element WPM medium.The plantlets were transplanted to soil after acclimatization.2.Establishment the Gentetie transformation System of Zanthoxylum piperitum DC var. inerme Makino mediated via Agrobacterium tumefaciensThe genetic transformation system of Zanthoxylum piperitum DC vat.inerme Makino was optimized by using the cotyledon and hypocotyl as the receptors mediated with Agrobacterium tumefaciens.The content of kanamycin（Kan） selection pressures were determined as 30 mg·L^-1 for shoot regeneration of petioles and stems.To monitor the induction rate of Kanamycin-resistant callus,the optimal parameters （such as pre-culture time,bacterium concentration,infection time,co-culture time.） for A.tumefaciens-mediated transformation of Z.piperitum DC van inernme Makino were examined.The results showed that 3 days’ pre-culture,10 min Agrobacterium(OD₆₀₀ about 0.5-0.7) infection with 100μmol·L^-1 acetosyringone（AS）,and 3 days’ co-culture would lead to an increase in transformation efficiency.3 Detection of transgenic Z.piperitum DC var.inerme Makino plantsThe binary expression element with ipt gene is transformed to Z piperitum DC var.inerme Makino via Agrobacterium mediated method.The results showed that many transformants showed blue when they were dyed and many also showed positive with PCR checking.All of these results prove that aimed gene has been transformed into Z piperitum DC var.inerme Makino.The expression of ipt gene has effectively promoted the adventive bud induction.Z.piperitum DC vat.inerme Makino with ipt gene could produce a great many branches and could easily be propagated,but rhizogenesis of transformants was restrained.更多还原