Identification Of NF-Y Gene Family In Apple And Investigation On The Function Of MdNF-YA8 In Response To Salt Stress

Nuclear factor Y（NF-Y）is a kind of transcription factor widespread in eukaryotes.It is also known as CCAAT box binding factor（CBF）,which can specifically recognize and bind with the CCAAT frame in the promoter region of target genes to regulate their expressions.In recent years,it has been found that NF-Ys play important roles in plant growth and in responses to abiotic stress.However,reports on the identification and functional analysis of NF-Y gene family in apple are still limited up to now.In 2019,Chinese scientists completed the whole genome sequencing of the apple strain of’Hanfu’,which provide a high-quality basis for apple bioinformatics analysis.In this study,11 MdNF-YAs,23 MdNF-YBs and 13MdNF-YCs were identified from the’Hanfu’apple genome and their responses under different stress conditions were also analyzed.In addition,the MdNF-YA8 gene was found to be significantly up-regulated induced by salt stress,and its resistance to salt stress was further analyzed by constructing transgenic Arabidopsis thaliana and apple callus.The main findings are as follows:（1）Identification of NF-Y gene family in apple.47 members of NF-Y family were identified from apple genome database using bioinformatics technology,including 11MdNF-YAs,23 MdNF-YBs and 13 MdNF-YCs,which were distributed on 17 chromosomes.Conservative domain analysis showed that all NF-YAs of apple contained CBF＿NF-YA domain,while all MdNF-YBs and MdNF-YCs subfamily members contain CBFD＿NFYB＿HMF conserved domain.By analyzing the conserved motif and gene structure,it is found that the members of the same subfamily have similar conserved motif and gene structure.（2）Analysis of cis-acting elements of MdNF-Ys promoter region and the stress responses of MdNF-Ys.The promoter sequences of about 1.5 kb upstream sequences of all MdNF-Ys member genes were predicted and analyzed.It was found that most of the promoter regions of MdNF-Ys contained elements associated with stress responses,such as drought,low temperature,abscisic acid and ethylene response elements.According to the analysis results of promoter elements,13 MdNF-Ys containing stress response related elements in the promoter region were selected to detect the changes of gene expression under different stress conditions,and it was found that the expression of seven MdNF-Ys genes was up-regulated in apple roots under NaCl stress,while the expression of MdNF-YA8 was the highest in roots,which was up-regulated by 21 times.After PEG simulated osmotic treatment,the expression of six MdNF-Ys were also significantly up-regulated in which the expression of MdNF-YA4and MdNF-YB13 in roots were more than 20 folds compared with control.Under exogenous ABA treatment,the expression of more than half of MdNF-Ys was significantly up-regulated,and MdNF-YB13 was up-regulated more than 50 times in roots.These results showed that most MdNF-Ys were up-regulated by multiple stresses.（3）Expression pattern analysis of MdNF-YA8 gene in apple.MdNF-YA8 is located in the nucleus and mainly expresses in apple roots,leaves and flowers.The 2 kb sequence upstream of MdNF-YA8 was cloned and recombined into a plant expression vector to drive the expression of reporter gene GUS,and then transformed into wild-type Arabidopsis and obtained a homozygous strain.Subsequently,the transgenic seedlings of one week old were treated in NaCl solution and stained with GUS.The results showed that the expression of GUS in the transgenic lines was significantly enhanced after salt stress treatment,further demonstrating that the expression of MdNF-YA8 was induced by salt stress.（4）Recombinant expression of MdNF-YA8 improved the salt tolerance of yeast cells.By using yeast recombinant expression of MdNF-YA8 with the transformed empty plasmid as the control,it was found that under salt stress,the yeast colonies overexpressing MdNF-YA8 grew significantly faster than the control;At the same time,overexpression of MdNF-YA8 in NaCl liquid medium also significantly improved the salt tolerance of the yeast.（5）Overexpression of MdNF-YA8 in Arabidopsis thaliana enhances the salt tolerance.A homozygous Arabidopsis thaliana strain overexpressing MdNF-YA8 was obtained by inflorescence staining.Under salt stress,the seed germination rate of the transgenic Arabidopsis thaliana was significantly higher than that of the wild type,and the root length of the transgenic seedlings was significantly longer than that of the wild type.By irrigating 250m M NaCl of Arabidopsis thaliana at four weeks of seedling age,it was found that the growth of the transgenic strains was significantly better than that of the wild type,the proline content of the OE strain was higher than that of the WT strain,and the MDA content was lower than that of the WT strain.Further detection of H₂O₂and O₂^-content in WT and OE strains under high salinity stress showed that the active oxygen species content in OE strains was significantly lower than that of the WT strain.In addition,under high salt stress,the activities of antioxidant enzymes in transgenic Arabidopsis plants were also significantly higher than those in wild type.（6）Overexpression of MdNF-YA8 increased salt tolerance of apple callus.Under normal conditions,the growth of WT and OE apple callus showed no significant difference,while under salt stress treatment,the volume of callus of OE strains was significantly greater than that of the WT strain.It was also found that the cell membrane permeability of OE strain was lower than that of WT under salt stress.Further analysis showed that OE strain had higher antioxidant enzyme activity.In addition,qRT-PCR showed that the expression of SOS pathway related genes（MdSOS1,MdSOS2,MdSOS3 and MdNHX1）were up-regulated under175 m M NaCl stress.