转MhGlu基因烟草灰霉病抗性及光合特性研究(英文)

Overexpression of MhGlu Enhances Botrytis cinerea Resistance in Tobacco

以湖北海棠盆栽及组培苗叶片为材料,经NaCl、PEG-6000及4℃下ABA处理后,通过RT-PCR技术克隆了湖北海棠β-1,3-葡聚糖酶基因MhGlu;构建MhGlu基因的植物表达载体,通过农杆菌介导法将MhGlu基因转入烟草中,并通过PCR和RT-PCR检测,成功获得了4个转基因株系T6、T8、T11和T18;以转基因烟草株系T6及T8和非转基因对照植株为材料,对MhGlu基因的功能进行了进一步分析。结果显示:(1)半定量qRT-PCR显示,NaCl、PEG-6000及4℃下ABA处理均可以诱导湖北海棠盆栽及组培苗叶片MhGlu基因的表达;NaCl和PEG-6000处理48h内MhGlu基因的表达随处理时间延长逐渐增强,4℃下ABA处理的MhGlu基因表达量在4h时开始上调,12h时略降低,48h时又达到最大。(2)半定量RT-PCR检测转基因烟草植株几个病程相关基因PRs的表达量,表明过表达的MhGlu基因诱导并增强了烟草病程相关基因NtPR1、NtPR3和NtPR5的表达。(3)用灰霉病侵染烟草叶片,转基因烟草株系T6、T8均表现出较强的抗灰霉病特性。(4)测定烟草植株光合特性参数,转MhGlu基因烟草株系的净光合速率(Pn)、蒸腾速率(Tr)和气孔导度(Gs)较对照组均显著提高,且T8的净光合速率和蒸腾速率均显著高于T6,而T8与T6的气孔导度差异不显著。MhGlu基因在烟草中的过量表达能诱导病程相关基因PRs的表达,激活了烟草的光合特性保护机制,提高了转MhGlu基因烟草植株的灰霉病抗性。

dIn our study,MhGlu,a β-1,3-glucanases gene of Malus hupehensis was cloned from leaves of M. hupenhensis treated with NaC1,PEG-6000 and ABA at 4 ℃ by RT-PCR. A plant binary expression vector of MhGlu was constructed. The vector was transformed into tobacco through Agrobacterium-mediated method. PCR and RT-PCR results showed that four transgenic tobacco lines T6, TS,Tll and T18 were obtained. The function of MhGlu was further analyzed with the transgenic tobacco lines T6, T8 and wild type tobacco plants （WT）. Results showed that： （1）The expression of the gene in M. hupehensis was monitoredin a 48 h course after treated with NaC1,PEG-6000 and ABA at 4 ℃, respectively. Semi-quantitative RT- PCR revealed that expression of MhGlu was induced by NaC1,PEG-6000 and ABA at 4 ℃ treatment in both potted seedlings and seedlings cultured in vitro. Treated by NaC1 and PEG-6000, the expression of MhGlu gene increased gradually over time; while ABA treatment at 4 ℃ up-regulated MhGlu 4 h post treatment,the expression decline at 12 h,then reached to maximum at 48 h. （2）Compared with the WT plants, several pathogenesis-related genes （PRs） in tobacco （NtPR1,NtPR2, and NtPR3） were up-regulated in tobacco plants with overexpression of MhGlu, detected by semi-quantitative RT-PCR. （3）Being in- fected with Botrvtis cinerea . （4）Overexpression MhGlu appeared to improve the photosynthetic characteristics of transgenic tobacco. The net photosynthetic rate （pn）, the transpiration rate （Tr） and stomatal conductance （G,） of transgenic plants were increased compared with that of controls, and the difference was significant. The T8 and T6 of T8 were significantly higher than that of T6, while the difference of Gs between T8 and T6 was not significant. Overall, the overexpression of MhGlu can induce the resistanceto B. cinerea of tobacco.