【作者】 刘茜；

【导师】 赵天永；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 纹枯病是由立枯丝核菌引起的一类病害,严重危害了我国的玉米生产。目前,尚无优良的抗纹枯病的玉米遗传种质。本研究利用遗传转化的方法将3个与抗病相关的外源基因（番茄转录因子基因Pti4,烟草β-1,3-葡聚糖酶基因NtGlu以及萝卜抗菌肽基因Rs-AFP2）转入玉米,以期获得遗传稳定的抗病株系。本研究以玉米HiII或A188的幼胚作为受体材料,分别利用基因枪转化和农杆菌介导两种方法,将带有NtGlu基因的超表达载体以及带有Pti4和Rs-AFP2基因的共表达载体分别进行玉米转化。两个载体均以bar基因作为筛选标记。HiII型幼胚经基因枪转化共得到4个超表达NtGlu的株系以及2个共表达Pti4和Rs-AFP2的株系。每代转基因株系均与自交系郑58进行回交。目前已得到T₄代种子,并用于抗病性鉴定。A188型幼胚经农杆菌转化最终得到了2个超表达NtGlu的株系,目前已得到T₂代种子。对T₄代转基因植株进行鉴定,结果表明外源基因在转基因玉米后代中得到稳定遗传和有效表达。用0.02%的Basta溶液涂抹玉米叶片后发现,与对照相比,转基因植株具有明显的除草剂抗性,且抗感分离比约为1:1。对离体叶片进行纹枯病菌接种实验,并统计接菌3天后的相对病斑面积以及相对电导率。结果证明,超表达NtGlu的株系以及共表达Pti4和Rs-AFP2的株系均对玉米纹枯病有一定的抗性。同时,NtGlu转化株系中β-1,3-葡聚糖酶的相对酶活比野生型株系显著升高。更多还原

【Abstract】 Sheath blight is one of the maize common fungal diseases caused by Rhizoctonia solani and threatens maize production in China.There is no known germplasm that are resistant to sheath blight that can be used for classic breeding.In this study,three exogenous defense-related genes including tomato（Lycopersicum esculentum）transcription factor Pti4 gene,tobacco（Nicotiana tabacum）β-1,3-glucanase gene NtGlu and radish（Raphanus sativus L）antifungal peptide gene Rs-AFP2 were transformed into maize.The transgenic maize plants and its progenies were characterized for the transgene integration,expression,transmission and the disease resistance.Immature embryos of maize HiII or A188 are used for maize transformation.Both gene particle bombardment and agrobacterium-mediated transformation method were applied in our research.The NtGlu expression vector and Pti4-Rs-AFP2 co-expression vector with Bar gene serves as a selection marker were successfully transformed into maize.Four NtGlu transgenic lines and two co-expression Pti4 and Rs-AFP2 transgenic lines were obtained using gene particle bombardment transformation with HiII.Every generation of transgenic plants were backcrossed to Zheng58 and T₄ generation plants were evaluated for its blight sheath disease resistance.Two NtGlu transgenic lines were obtained using agrobacterium-mediated transformation method with A188.T₀ plants were backcrossed to Zheng58 and T₂ generation were harvested.Molecular characterization of the T₄ lines demonstrated that the foreign genes have been successfully transmitted to the forth generation and stably expressed.The T₄ plants were evaluated for the herbicide basta resistance by staining with 0.02%Basta.The results showed that the ratio of resistance plants to non-resistance plants is about 1:1.The detached leaves of basta resistance plants and WT plants were inoculated with Rhizoctonia solani and the relative lesion area and electric conductivity were measured.The results showed that both NtGlu expressing plants and co-expressing Pti4 and Rs-AFP2 plants had significantly higher resistance to maize sheath blight compared with WT control.Furthermore,the relative activity ofβ-1,3-glucanase in NtGlu transgenic lines is significantly increased than wild type.更多还原