Cloning Foxtail Millet Stress Resistance Transcription Factor Gene SiNAC110 And SiMYB148 For Function Identification

Soil salinity,drought and low nitrogen have been become potential threat to plant growth and crops yield.Foxtail millet(Setaria italic L.)that was famed as a model crop for stress tolerance.Foxtail millet(Setaria italic L.)is the oldest cultivated crops.Compared to other crops,foxtail millet(Setaria italic L.)has strong stress tolerance,small genome,and genetic diversity,which may enhance the ability to adapt to abiotic stress.However,the function and mechanism of resistance foxtail millet(Setaria italic L.)gene remains elusive.Currently,Foxtail millet(Setaria italic L.),whole genome sequencing has been completed,which will provide an excellent platform for foxtail millet(Setaria italic L.)functional verification.On the basis of foxtail millet(Setaria italic L.)transcriptome sequencing,choosing drought upregulated SiNAC110 transcription factors and low nitrogen upregulated SiMYB148 transcription factor,and then sequence analysis of these two genesgene structure,Arabidopsis protoplasts to identify the position of SiNAC110 and SiMYB148.Real-time fluorescence quantitative PCR to analysis expression patterns of SiNAC110 and SiMYB148 under different abiotic stresses.Phenotypic analysis SiNAC110 overexpression lines under drought and high salt stress and statistical analysis germination rate,root length,root area,fresh weight,dry weight.Real-time quantitative PCR method for analyzing drought and salt stress related downstream gene.Finally under hormone ABA treatment,verified whether SiNAC110 dependence ABA signaling pathway.Phenotypic analysis SiMYB148 transgenic rice using hydroponic culture experiments under low nitrogen stress.Statistical analyzes were performed on root length,root surface area,fresh weight,plant height and nitrogen content.Germination phenotype analysis of SiMYB148 transgenic rice under high salt stress.Final under ABA treatment,verified SiNAC110 transgenic rice whether dependence ABA signaling pathway or not.1.The transcription factor gene SiNAC110(1)SiNAC110 gene sequence analysis,SiNAC110 had the total length of 1101 bp,encoding 366 amino acids,the molecular weight of 40.808 KD and isoelectric point of 6.31.There was a conserved NAM domain between 11~139 amino acids.The SiNAC110 had a highest homology gene with SiNAC105.(2)SiNAC110 protein subcellular localization resultsrevealed that it was localized in the nucleus.(3)The gene expression results indicated SiNAC110 could be induced expression by drought and high salt,the expression level was more than 20 fold than before.(4)Overexpression of SiNAC110 gene in Arabidopsis and for functional verification,SiNAC110 transgenic lines in the root length,root surface area,fresh weight,fry weight and germination rates increased more rapidly with MS plus PEG and NaCl medium than WT.(5)The activity of the key enzyme involved in proline synthesis and Na+transport was higher,indicating that higher level of proline synthesis and preventing rapid accumulate of Na+ in the cytoplasm contributed to enhanced drought and high salt resistance of SiNAC110 transgenic plants.(6)Verified SiNAC110 whether the ABA-dependent signaling pathways,there are no significant phenotypic difference in MS plus ABA medium.The expression level of ABA-releted gene did not reach increased.So overexpression of SiNAC110 could simultaneously improved drought and high salt resistance with independent ABA pathway.2.The transcription factor gene SiMYB148(1)SiMYB148 gene sequence analysis,SiMYB148 had the total length of 912 bp,encoding 302 amino acids,isoelectric point of 5.65,and the molecular weight of 33452.8 KD.The gene structure analysis SiMYB148 belong MYB-R2R3 type transcription factor.(2)Protein subcellular localization of SiMYB148 revealed that it localized in the nucleus.(3)The gene expression profile indicated SiMYB148 could be induced by dehydration,high salinity stresses,low nitrogen and ABA.(4)Overexpression of SiMYB148 gene in rice and for functional verification,The results showed that under low nitrogen and drought stress,overexpression line had longer strain root,larger root surface area,heavier fresh weight,higher height and lateral root than the control.Nitrogen content measure showed that overexpression lines both aboveground and underground parts have higher nitrogen content than the control.Under high salt stress,the germination rate statistics showed that overexpression lines increased more rapidly than control plants.(5)Verified SiMYB148 whether dependent the ABA signaling pathways,indicated that overexpression SiMYB148 in rice is high sensitive to ABA.It was proved SiMYB148 by the ABA signaling pathway to response to abiotic stress.