Transformation Of Rice (Oryza Sativa L.) With Anti-apoptotic Genes Confers Resistance To Sheath Blight And Seedling Rot

In order to obtain new germplasms having durable, broad-spectrum resistance to diseases caused by necrotrophic pathogens and tolerance to abiotic stresses during rice production, baculovirus anti-apoptotic genes Op-iap and p35 were transferred, for the first time to our knowledge, into Japonica rice by Agrobacterium-mediated transformation.Baculovirus anti-apoptotic genes Op-iap and p35 were cloned from pIEl-4opiap and pIEl-4p35 using PCR with three pairs of primers designed according to the encoding region sequences. The encoding region of Op-iap with 807 bp in length encodes a 30 kDa protein which can block some kinds of apoptosis. The encoding region of p35 is 900 bp in length and encodes a 35 kDa protein which can also block some kinds of apoptosis in animal and plant cells. Three expression vectors were constructed by inserting Op-iap and p35 into binary vector pCAMBIA1301 (pl301 for short) singly and in combination. The expression vectors, including pl301-Op-iap, pl301-p35, pl301-iap-p35 and pl301 itself (as CK), were proven to be constructed correctly by PCR analysis, enzyme digesting analysis and sequence analsis.Totally 485 transgenic rice plants were obtained by transferring the genes into three rice cultivars (Oryza sativa L.subsp. japonica), Chinese cultivars Zhonghua 8, Zhonghua 10, and Japanese cultivar Aichiasahi, using Agrobacterium-mediated transformation. PCR, Southern-blot, Northern-blot and RT-PCR analyses showed that the target genes were integrated into transgenic rice genome and that completed mRNA strand was produced by transcription. Both Op-iap and p35 showed a Mendelian segregation ratio and a tight linkage heritance with selection marker gene hpt in T1 generation of transgenic rice.Average disease rating and disease index of rice sheath blight were investigated after inoculating with Rhizoctonia solani. We extracted DNA from the surrounding tissue of the lesion and electrophoresed it in 2% agarose gel to observe DNA fragmentation which is the sign of apoptosis. The results of observation, calculation and statistic analysis indicated that foreign anti-apoptotic genes confer transgenic rice plants the ability to block apoptotic cell death as induced by the necrotrophic fungal pathogen, leading to a significant increase in the resistance to sheath blight.Expression of anti-apoptotic genes blocked apoptotic cell death induced by necrotrophic fungal pathogen, and conferred rice seedlings of Tl generation an increased resistance to seedling rot caused by Rhizoctonia solani when growing in the environment of low temperature, low solar radiation, and short daylight period. The resistance difference between transgenic plants and controls is statistically significant.Our observations showed that anti-apoptotic genes did not affect disease resistance to rice blast when inoculating with compatible and incompatible race of Magnaporthe grisea. Other growth and development processes, including reproduction, seed germination, and morphogenesis, were not impacted by anti-apoptotic genes either.