蛋白激发子基因pemG1转化三生烟及其对TMV抗性的提高

Expression of Protein Elicitor-Encoding Gene pemG1 in Tobacco (Nicotiana tobacum cv. Samsun NN) Plants and Enhancement of Resistance to TMV

通过根癌农杆菌(Agrobactrium tumefaciens)介导转化法,将含有稻瘟菌蛋白激发子基因pemG1的植物表达载体pCAMBIA2300-Ubi-pemG1-Ocs转化三生烟(Nicotiana tobacum cv.SamsunNN),获得了转基因植株。用PCR检测抗卡那霉素烟苗确认阳性转化株,用Southern、Northern和Western杂交进一步证实了pemG1基因的整合、转录和表达。对T2代转基因阳性株进行烟草花叶病毒(Tobacco Mosaic Virus)接种试验。接种5d后发现,与非转基因对照相比,表达pemG1的烟草叶片枯斑数量减少,表明稻瘟菌蛋白激发子基因pemG1的表达提高了转基因烟草对TMV的抗性。

Protein elicitors are important signal molecules that trigger plants disease resistance. Defence responses will be induced once the elicitors are recognized by acceptors in plants. It is revealed that elicitor protein PemGl from Magnaporthe grisea is able to increase hydrogen peroxide content of tobacco suspension cells. To study PemGl＇s functions in plants, pemG1 gene was trans- ferred into tobacco in the study. For this, plant expression vector pCAMB1A2300-Ubi-pemG1-Oc harboring elicitor-encoding gene pemG1 from Magnaporthe grisea was constructed. The maize ubiquitin promoter/octopine synthase terminator system and kanamycin-resistant gene nptll （neomycin phosphotransfers Ⅱ） were used for constitutive expression systems. The vector was then introduced into Agrobacterium tumefaciens （strain AGL-1） by freeze-thaw method. Tobacco （Nicotiana tobacum cv. Samsun NN） primary transformants were produced by leaf disc transformation. The kanamycin-resistant regenerated plants were confirmed to be electropositive by PCR. Integration and expression of the pemGl gene were further confirmed by Southern blotting and Western blotting, respectively. Then, transgenic tobacco plants of T2 generation were inoculated with Tobacco Mosaic Virus （TMV） at two different virus concentration. In comparison with TMV-infected wild-type SNN plants, PemGl-expressed plants displayed reduced hypersensitive-response lesions in both treatments. Furthermore, accumulation level of pemG1 steady-state transcripts was examined at 24 h after inoculation. The results indicated that the reduction of lesions corresponded to the accumulation of pemG1 Steady-state transcripts as monitored by Northern blotting analysis. All these indicated that the expression of pemG1 in tobacco plants improved the resistance to TMV.