The Transcriptome Analyses Of Daylily (Hemerocallis ’Autumn Red’) Under Seawater Stress

Daylilies(Hemerocallis spp.& cvs.),one of the three major perennials in the world,enjoy high ornamental value and adaptability,are widely used in road,park,green space,home gardening and other fields.China is the natural distribution center of Hemerocallis spp.in the world.Daylily has a cultivation history of 3000 years.Daylily is also known as the "Mother Flower of China" since ancient times.However,soil salinization in coastal areas of East China not only affects the ornamental quality of daylilies,but also seriously restricts the promotion and application of daylilies in this area.Therefore,it is of great significance to breed daylilies with high salt resistance.In this study,high-throughput sequencing technology was used to sequence the 3-generation + 2-generation transcriptome of Hemerocallis ’Autumn Red’under seawater(salt)stress.The differentially expressed genes of daylilies under salt stress were analyzed and compared,and the genes involved in salt stress response were screened for GO classification and KEGG enrichment analysis.The bZIP gene expression of daylilies under salt stress was analyzed to reveal the molecular mechanism of daylilies salt tolerance from the transcriptome level,in order to provide scientific basis for the molecular biology research and molecular breeding of daylilies salt tolerance.The main research results are as follows:1.Using the combination of Pac Bio Sequel full-length transcriptome sequencing technology and Illumina sequencing technology,transcriptome sequencing analysis was performed on Hemerocallis ’Autumn Red’ treated with seawater for 0h,3h,6h,12 h,24h and72 h.The results showed that a total of 19550 genes were detected,with a total sequence length of 31440008 bp and an average length of 1609 bp.These data can not only reflect the changes of daylilies gene transcription level under salt stress,but also provide data reference for further research on the molecular regulation mechanism of daylilies resistance to salt.2.Differential screening was performed according to the expression levels of protein-coding genes in different samples.In order to understand the dynamic changes of gene expression of daylilies under salt stress with treatment time,we focused on five different groups,which were 3h-VS-0h,6h-VS-0h,12h-VS-0h,24h-VS-0h,and 72h-VS-0h.When the screening condition was set as FC > and P < 0.05,the number of differential protein coding genes detected were 11969,11327,11725,13142 and 13027,respectively.3.GO enrichment analysis of differentially expressed genes in 6h-VS-0h and24h-VS-0h comparison groups showed that differentially expressed genes in these two comparison groups were mainly concentrated in the oxidation-reduction process classification of biological processes;Enzymatic catalytic activity,oxidoreductase activity,hydrolase activity,cofactor binding classification of molecular function.It is worth noting that the differentially expressed genes were mainly concentrated in the cell wall and outer encapsulation structure classification of cell components after 6h of salt stress treatment.With the extension of treatment time,there was no enrichment of differentially expressed genes in cell components after 24 h of stress treatment.Therefore,it is speculated that ROS-mediated cell wall loosening or hardening during the early stage of salt stress can enable plant cells to withstand the turgor changes under osmotic stress,thus improving the salt tolerance of plants.4.KEGG enrichment analysis showed that DEGs in the two comparison groups were significantly enriched in starch and sucrose metabolic pathways,amino and nucleotide sugar metabolic pathways,and phenylpropane-like metabolic pathways,suggesting that daylilies root responded to salt stress through a variety of metabolic pathways,and these DEGs were involved in the regulation of the above metabolic pathways.5.By comparing the bZIP family relationships between daylilies and model plants rice and Arabidopsis thaliana,we divided the related bZIP genes into eight subgroups,and then predicted the protein secondary structure of each subgroup.It was found that motif2 and motif7 appeared only in the D subfamily,showing high conservatism.According to the conserved motif distribution of daylilies bZIP protein,it was speculated that motif1 and motif10 were important components of daylilies bZIP protein.The analysis showed that the differences in the conserved motifs were small among the family members with high homology,which was consistent with the phylogenetic relationships of the proteins analyzed,and verified the reliability of the phylogenetic relationships.6.Through the process of salt stress after daylilies bZIP gene expression is analyzed,according to the function of the evolutionary tree related theories,similar to C subtribe HfbZIP8 of arabidopsis thaliana and rice bZIP genes are involved in resistance to salt stress physiological process,and its expression rule is consistent with the experimental results,Therefore,HfbZIP8 may be a bZIP gene that plays a key role in the physiological processes related to salt stress resistance in daylilies.However,the expression of HfbZIP27 and HfbZIP28 in G subgroup is always low.A large number of literatures have confirmed that AtbZIP24,a close Arabidopsis bZIP gene,is an important negative regulator of plant salt stress response.Therefore,if RNA interference test is used to silence its expression,it may improve the salt tolerance of plants.