Analysis And Identification Of Stress Resistance-related Genes Of Soybean NF-YC Transcription Factor And Foxtail Millet CDPK Protein Kinase Families

Abiotic stresses such as drought,high-salt,high-temperature and low-temperature,severely affected the growth and development of crops.Therefore,it is meaningful for discovering the stress resistance genes and studying mechanism of crop plant stress resistances.Numerous studies revealed A and B subunits of NF-Y transcription factors involved in resisting abiotic stresses,however,many studies about C subunit of NF-Y transcription factors focused on affecting plant photoperiod and promoting plant flowering.But recent research revealed C subunit familiy(NF-YC)of NF-Y transcription factor played an important role in resisting abiotic stresses.Plant CDPK protein kinase plays an important role in plant resistance to abiotic stress,and no special articles written about foxtail millet(Setaria italica)CDPKs systematical analyses.Soybean(Glycine max L.Merr.)and foxtail millet are main oilseed crops and gain crops in China.Our research revealed soybean NF-YCs and foxtail millet CDPKs played important roles in resisting abiotic stresses.The main results of the research were shown below:1.We collected the information about NF-YC families by scanning for genomic data of soybean,Arabidopsis,rice,foxtail millet and maize.Finally,after removal of the repetitive sequences,87 non-redundant soybean NF-YC proteins were identified and classified into four clusters according to their phylogenetic relationship by using MEGA5.0 software.Exon and intron structures and the conserved domains of soybean NF-YCs were analyzed by using GSDS online tool and DNAMAN software,respectively.25 soybean NF-YC genes were mapped on 14 chromosomes by using Map Draw software.Promoter analysis showed 25 soybean NF-YCs member genes contained cis-acting elements that related to abiotic stresses.Expression profile analysis showed most soybean NF-YCs member genes could be induced to up-regulate by drought and salt.A few members could be induced by ABA and low-temperature.q RT-PCR analysis showed among 25 soybean NF-YCs,Gm NF-YC9 could be induced by drought and salt,and the relative expression levels of Gm NF-YC9 were highest than that of others under drought and salt stress.Therefore,Gm NF-YC9 were selected for the further study.Gm NF-YC9 protein located in cell membrane and cell nucleus,and its nuclear localization could be induced by stress.The phenotypic characterization analyses of transgenic Arabidopsis and soybean hairy root plants showed overexpression of Gm NF-YC9 could enhance the tolerance to drought and salt stresses in transgenic plants.RNA-seq data analysis showed overexpression of Gm NF-YC9 in transgenic Arabidopsis and soybean hairy root plants could improve the expression of stress responsive genes.2.Screening and identification of the interacted proteins of Gm NF-YC9.The interacted proteins of Gm NF-YC9 were screened by using the method of yeast two hybrid.Among the candidate proteins,B-subunit protein Gm NF-YB24 were selected for further study.The interactions between Gm NF-YC9 and Gm NF-YB24 were further verified by yeast two hybrid,Bi FC and GST-pulldown technologies.Gm NF-YB24 protein located in cell nucleus,cytoplasm and cell membrane.Overexpression of Gm NF-YB24 in soybean hairy roots could also enhance the drought-and salt-tolerances of transgenic soybean hairy roots.Our research revealed soybean Gm NF-YC9 protein complexity in plant resistance to abiotic stress by analyzing functions of candidate protein soybean Gm NF-YB24.3.Following the analysis method mentioned,we analyzed the CDPK protein kinase families in different plant species.94 CDPKs were identified by global sequence analysis and divided into 4 subtribes by neighbor-joining phylogenetic tree analysis.Gene structure analysis showed most foxtail millet CDPKs had introns.A large number of cis-acting elements that related to abiotic stresses were found in promoters of foxtail millet CDPKs.q RT-PCR analysis showed most foxtail millet CDPKs were induced by drought and ABA.These result suggested soybean NF-YCs and foxtail millet CDPKs involved in plant resistance to abiotic stresses.While,Si CDPK24 had the highest expression than others at 12 h of drought treatment.Therefore,Si CDPK24 were selected for the further study.Foxtail millet Si CDPK24 protein located in cell nucleus and cell membrane.The phenotypic characterization analyses of transgenic Arabidopsis plants showed overexpression of Si CDPK24 gene in Arabidopsis could enhance the tolerances of transgenic Arabidopsis plant to drought stress.The expression patterns of stress related genes were analyzed in transgenic Si CDPK24 Arabidopsis plants and WT by q RT-PCR.The result showed DREB2 A,CBF3,RD29 A,RD29B,RD22,COR47,COR15 A,KIN1 and LEA1 had significantly higher expression levels than that of WT after drought treatment.Further analysis revealed Si CDPK24 gene could improve the transcriptions of target genes(RD29A,RD29 B,RD22,COR47,COR15 A,KIN1 and LEA1)by enhancing activities of the two DREB transcription factor DREB2 A and CBF3.In this study,we used Arabidopsis and soybean as research objects and demonstrated soybean Gm NF-YC9 and foxtail millet Si CDPK24 genes could enhance plant stress tolerances by improving the expressions of stress responsive genes,and revealed C subunit protein of plant NF-Y transcription factor play an important role in plant resistance to abiotic stress,and analyzed the regulatory mechanism of plant stress resistance of soybean Gm NF-YC9 and foxtail millet Si CDPK24 genes.These results offered the theoretical foundation for the breeding of soybean resistance varieties.