Abiotic Stress Resistance Evaluation Of Rice Co-overexpression Of OsbZIP46CA1 And SAPK6

Rice(Oryza sativa L.),is one of the most important food crops in the world,and it is a staple food for more than half of the world's population.However,rice production is facing challenges due to the global climate change,including drought which has severely affected the world economy and triggered food security issues.Therefore,the genetic improvement strategy related to drought resistance in rice varieties is one of the leading research themes.Drought resistance is controled by multiple genes,and thus,a multi-gene used in genetic engineering strategy is theoretically useful for improving drought resistant ability in rice.However,the experimental evidence for such strategy is still lacking.In this study,drought-responsive genes were isolated from rice and then were assembled through multiple-round in vivo site-specific assembly(MISSA)system,and the constructs were introduced into the rice cultivar KY131 via Agrobacterium-mediated transformation.Overexpression transgenic lines of the multi-gene and corresponding single-gene constructs containing a single copy were identified by analyzing the transcription level and copy number of the transgene in T1 generation.The overexpression transgenic lines and negative transgenic plants(KY131-N)were used for pre-evaluation of drought resistant ability in the field and for further experiments.During the pre-evaluation,co-overexpression transgenic lines of the two genes OsbZIP46CA1(encoding a constitutively active form of the bZIP transcription factor OsbZIP46)and SAPK6(encoding a sucrose nonfermenting 1 [SNF1]-related protein kinase SnRK)remarkably enhanced drought resistance compared with the single-gene transgenic lines(CA1-OE and SAPK6-OE)and KY131-N based on visual evaluation of the leaf death and seed-setting at the reproductive stage.The co-overexpression transgenic lines(XL22),compared with the single-gene overexpression lines and KY131-N,were hypersensitive to ABA.This suggested that the co-overexpression of OsbZIP46CA1 and SAPK6 may have an additive effect on the ABA sensitivity of rice.The XL22,CA1-OE,SAPK6-OE overexpression transgenic lines and KY131-N in drought resistance in the field using agronomical traits were further evaluated.The results indicated that XL22 exhibited higher yield,biomass,spikelet numbers and grain numbers under moderate drought stress conditions compared with the single-gene overexpression lines and KY131-N.The results on water loss rate and drought stress survival rate of the transgenic seedlings alsosupported the enhanced drought resistant ability of XL22.Furthermore,expression profile using a RNA-Seq analysis revealed that many genes involving in stress responses were specifically up-regulated in the droughttreated XL22 lines and some of the stress-related genes activated in CA1-OE and SAPK6-OEwere distinct,which could partially explain the different performances of these lines in response to drought resistance.In addition,tolerance to heat(42oC)and cold(4oC)stresses of the overexpression transgenic lines and KY131-N were tested at the seedling stage.Results revealed that,compared with the CA1-OE,SAPK6-OE,and KY131-N,XL22 lines showed a significantly higher survival rates in both heat and cold treatments.Besides,the malondialdehyde(MDA)content and the relative electrolyte leakage rate of XL22 lines were also significantly lower under the heat stress,indicating that XL22 lines have a low degree of membrane lipid peroxidation under heat stress and partically explained the enhanced heat resistance of XL22.These results demonstrated that overexpression of the two genes could enhance heat and cold resistant ability of rice.Taken together,the results from this study suggest that the multi-gene assembly in an appropriate combination may become a promising approach that can be possibly applied in genetic improvement of the stress resistance in rice.