| Rapeseed is one of the extremely important oil crops in the world,with rich nutritional value.It is a Brassica crop of the Brassicaceae family.Drought affects the growth and development,yield and seed quality of Brassica napus.The physiological characteristics of drought resistance that have evolved in plants also plays a major role in abiotic stress.Nuclear Factor Y（NF-Y）is a sequence-specific transcription factor containing nonspecific DNA-binding nucleosomes which was widespread in fungi,mammals,and plants.It consists of three different subunits-NF-YA,NF-YB,and NF-YC.NF-Y transcription factors play a very important role in plant response to drought stress.In this study,the Colombian wild-type Arabidopsis WT and rapeseed J9709 were used as background materials,the overexpression Arabidopsis material Bna A3.NF-YB8 and overexpressed transgenic rapeseed were obtained by genetic transformation.We observed and analyzed the WT and overexpression material Bna A3.NF-YB8 from gene function analysis and gene expression pattern analysis,experiments such as q RT-PCR verification,phenotype analysis of transgenic lines,determination of plant drought resistance,subcellular localization,promoter analysis,transcriptome analysis and yeast two-hybrid verification were mainly carried out in this study.The main conclusions are as follows:1.The full-length CDS sequence of Bna A3.NF-YB8 was cloned and constructed into a PEGAD fusion expression vector.Using the agrobacterium-mediated Arabidopsis protoplast transformation method,the observation results under a confocal microscope showed that the GFP fluorescent protein fused with Bna A3.NF-YB8 was localized to the nucleus,indicating that Bna A3.NF-YB8 functions mainly in the nucleus of plants.2.The 2 kb promoter sequence upstream of the Bna A3.NF-YB8 gene was inserted into the upstream of the GUS reporter gene of the p Cambia1305.1 vector,and the p Bna A3.NF-YB8:GUS expression vector was constructed to transform Arabidopsis.The spatiotemporal expression patterns and tissue-specific expression under abiotic stress were observed.It was found that under normal conditions,Bna A3.NF-YB8 was mainly expressed in the hypocotyl,young leaves,mature siliques peel,and the tip of the young siliques.It indicated that the expression sites of Bna A3.NF-YB8 gene were not the same in different stages,but from the complete growth stage,it was mainly expressed in hypocotyl,leaves and siliques.However,under abiotic stresses,it was mainly expressed in the hypocotyl and leaves,that is,abiotic stress altered the expression of p Bna A3.NF-YB8 in response to drought stress.3.The protein interacted with Bna A3.NF-YB8 were screened by the Brassica napus Genome website.The full-length coding sequence of the Bna A3.NF-YB8 gene was integrated into the bait vector p GBKT7,and the Bna A3.NF-YB8 protein was tested for toxicity and self-activation.The toxicity test was carried out on SD/-Trp selection medium,and it was found that Bna A3.NF-YB8 had no toxicity,and then self-activation verification was carried out on SD/-Trp-Leu selection medium.Compared with the positive control,Bna A3.NF-YB8 did not grow,indicating that it is not self-activating.The p GBKT7-YB8 protein was used as bait and the selected interacting proteins were screened on SD/-L-T and SD/-A-H-L-T+X-GAL-selection medium,It was found that the three NF-YCs proteins,Bna C8.NF-YC1 and Bna A7.NF-YC2,Bna C8.NF-YC9interacted with the Bna A3.NF-YB8 protein.4.The expression patterns of Bna A3.NF-YB8 gene under ABA,SNP,H2O2 and dehydration conditions were analyzed by q RT-PCR,and it was found that ABA,SNP,H2O2 and dehydration treatment could induce the up-regulated expression of Bna A3.NF-YB8.The analysis of its expression pattern under dehydration and its dependence on ABA,H2O2 and NO showed that the up-regulation of Bna A3.NF-YB8 under dehydration was independent of H2O2,NO and ABA.5.The phenotype observation of Bna A3.NF-YB8 overexpressed Arabidopsis under drought stress and salt stress found that the wilting degree of four overexpression lines OE-1,OE-2,OE-3 and OE-4 was slighter than that of WT and nf-yb8.After dehydration treatment,the overexpression lines could resume normal growth,while the WT and mutant nf-yb8 could not resume growth,and still showed wilting until death.Through the statistical analysis of the survival rate after drought and dehydration,it was found that the plant survival rate of overexpression lines was much higher than that of the WT and nf-yb8,it indicated that the Bna A3.NF-YB8 overexpressed Arabidopsis line had stronger drought tolerance than WT.However,in the phenotype observation of salt stress,it was found that the overexpression of Bna A3.NF-YB8 lines showed the same phenotype of leaf chlorosis and wilting as WT and nf-yb8,it indicated that overexpression of Bna A3.NF-YB8 did not enhance salt tolerance in transgenic Arabidopsis.6.Overexprssed lines and WT and nf-yb8 under drought stress were stained with DAB and NBT dyes.In the DAB staining experiment,the number of dark spots in the leaves of Bna A3.NF-YB8 overexpressed transgenic lines OE-1,OE-2,OE-3 and OE-4was significantly less than that of WT and mutant nf-yb8.The degree of coloration was significantly weaker than that of WT and nf-yb8,indicating that the H2O2 accumulation in leaves of overexpressed Arabidopsis was lower than that of WT and nf-yb8,showing more drought-tolerant characteristics;Similarly,in NBT staining experiment,the number of blue spots in the leaves of Bna A3.NF-YB8 overexpressed transgenic lines OE-1,OE-2,OE-3 and OE-4 was significantly less than that of WT and nf-yb8.The degree of plaque coloration was significantly weaker than WT and nf-yb8.It showed that the overexpressed Arabidopsis accumulated less reactive oxygen species and exhibited more drought-tolerant characteristics.7.The germination rate and green cotyledon rate of Bna A3.NF-YB8 overexpressed Arabidopsis lines were observed and counted under various abiotic stresses,It was found that under normal conditions,the germination rate and green cotyledon rate of WT,nf-yb8 and four overexpression transgenic lines OE-1,OE-2,OE-4 were similar.Under the treatments of 80 m M Na Cl,200 m M Mannitol,and 0.2μM ABA,the germination rate and green cotyledon rate of the overexpression lines were significantly higher than those of WT and nf-yb8,it indicated that the drought resistance of the overexpressed plants was stronger than that of the WT and nf-yb8.Root length measurement was performed under the same conditions.8.Transcriptome sequencing analysis found 1106 differentially expressed genes were obtained,including 603 up-regulated differentially expressed genes and 503 down-regulated differentially expressed genes.KEGG enrichment analysis showed that it was mainly enriched in metabolic pathways（ko01100）,synthesis of secondary metabolites（ko01110）,phytohormone signal transduction（ko04075）,alanine,aspartate and glutamate metabolism（ko00250）and other pathways,identified 824518 and 5 DEGs,respectively.Analysis of differentially expressed genes revealed many genes involved in drought stress response were up-regulated.These include genes NACs,MYB49,WRKY40,and LEA14,which play important roles in the response to drought stress.9.After genetic transformation of rapeseed,the overexpressed transgenic rapeseed OEYB8-1 was obtained.After drought treatment,it was found that compared with the control J9709,the degree of wilting of OEYB8-1 was slighter,the growth recovery after rehydration was better,which proved that overexpression of Bna A3.NF-YB8 enhanced the drought tolerance of transgenic rapeseed. |
