Study On Resistance To Sheath Blight For Transgenic GAFP Rice Lines

Sheath blight, which caused by Rhizoctonia solani Kiihn, is an important fungal disease in rice. It leads to huge yield loss and affects rice quality severely. Gastrodia antifungal protein (GAFP) isolated from traditional Chinese medicinal herb Gastrodia elata Bl. is a small protein with a broad spectrum of antifungal activity and it shows the strong resistance to rice diseases by many pathogenic fungi. It may be applied in plant antifungal genetic engineering.In this study, the gene encoding Gastrodia antifungal protein was introduced into two rice cultivars Xudao3(Japonica) and R6547(Indica) by Agrobacterium mediated transformation. The integration, transcription and expression of the gene were tested. Transgenic plants were grown in paddy field to study the resistance to sheath blight. The results are summarized as follows:1.Optimizing tissue culture system:(1) Optimization of the concentration of NaCIO solution and the sterilization time.The optimized sterilization is finally chosen. After removing the shell, the rice seeds are rinsed by70%C2H5OH for1-2minutes, immerged into25%NaClO solution for30minutes, finally, washed by distilled water.(2) The effect of adding ABA on rice callus inductionIn the culture of R6547, adding certain concentration of ABA solution can help to inhibit excessive growth of the germ, make callus more compact and enhance the induced rate of callus.2、Molecular detection of transgenic plantsPositive transgenic plants were detected by PCR method. The GAFP’gene integration was tested by Southern Blot analysis. The transgenic transcription was verified by RT-PCR. SG2gene relative expression was detected by Real-time RT-PCR. Genetic analysis showed that the transgenes were segregated by3:1in the progenies.3、Identification of resistance of T2generation in field(1) Identification of resistance to sheath blight for transgenic strains of Xudao3The resistance of progenies of Xudao3with5expression vectors was tested. Five vectors were G3, SG3, G4, SG4, SG2, respectively. The result showed that the resistance to rice sheath blight among all the T2generations with GAFP increased significantly compared with the control. The highest resistant transgenic line is the one by SG2vector, with2.03score alleviated in2011, a light-disease year.(2) Identification of resistance to sheath blight for transgenic strains of R6547The resistance of progenies of R6547with5expression vectors was tested. Five vectors are G1、G2、G4、SG3、SG4, respectively. The result showed that the resistance to rice sheath blight among all the T2generations with GAFP increased significantly compared with the control. The highest resistant transgenic line is the one by SG3vector, with2.21score alleviated in2011.(3)The correlation between the resistance and exogenous gene expressionThe result of Real-time RT-PCR revealed that the accumulation of SG2transcripts expression is related to the resistance level to rice sheath blight.