Functional Verification Of Heat Shock Transcription Factor HSF Of Rosa Chinensis

Rosa(Rosa Cvs),is a kind of perennial evergreen woody plant in the Rosaceae Family.It belongs to evergreen and semi-evergreen low shrubs.It blooms in all seasons,which has gorgeous colors and thousands posture.It plays an vital role in landscaping and deeply loved by people.It is also an important ornamental plant and one of the four fresh cut flower.When the temperature is higher than 35?,the leaves of the rose will be wilted,curled and burnt,which will seriously affect the high quality of the rose and reduce ornamental value.Heat Shock Transcription Factor(Hsfs)is an important transcription factor discovered in recent years.It is widely distributed in plant cells and plays a vital role to activate the expression of heat-resistant genes under heat shock.Therefore,studying the family of heat shock transcription factors in the rose is critical for improving the heat resistance of the rose.In this study,we identified HSF family members from the genomics of'Old Blush',and analyzed the chromosomal location,gene structure,conserved domains,and phylogenetic relationships of family members.The expression levels of HSFgenes in rose leaves under high temperature stress were analyzed by transcriptome sequencing,and the functions of two Hsf genes were studied.The main research results are as follows:1.A total of 18 HSF family gene members were identified from the 'moon moon powder'genome and named RcHsfl-RcHsf18.By constructing a phylogenetic tree with Arabidopsis AtHsf members,18 RcHsf members were divided into A,B and C categories,of which A had the most members,11 were followed by B,and 6 were C which is only one family member.Members of the RcHsf family of the rose are distributed on chromosomes 1-7 of the rose,and the RcHsf members distributed on chromosome 2 have the largest number of members.2?Domain analysis of members of the RcHsf family of the rose,18 family members contain highly conserved DBD domains and HR-A/B regions,The 11 HSF genes have NLS regions,including 9 A-type HSF genes,1 B-type HSF,and 1 C-type HSF;only some of the class A HSF members have an AHA domain.3.The expression of RcHsf family gene in leaves of high temperature stress was analyzed by expression profiling.11 unigene genes were differentially expressed,among which 10 unigene genes were significantly up-regulated under high temperature stress,5 were classified as Class A HSF,and 5 were classified as Class B HSF.The expression level of one unigene gene was significantly down-regulated and belonged to class A HSF.The expression of contig113972 in class A unigene gene is up-regulated under high temperature stress.The expression of conting12075 in the class B unigene gene increased most under high temperature stress.It can be concluded from the phylogenetic tree that contig 113972 corresponds to RcHsf16 and belongs to the HsfA2 subfamily,and contig12075 corresponds to RcHsfl,belonging to the HsfB2 subfamily.Therefore,the two unigene genes were selected for functional verification and named RcHsf1 and RcHsf164.The cDNA sequence of RcHsf1 and RcHsf16 was isolated from the rose.The cDNA of RcHsf1 located on chromosome 1 is 890 bp long,which contains 289 amino acids,isoelectric point of 8.71,and a molecular weight of 32119.07 KDa.RcHsf16 located on chromosome 7 has a cDNA length of 1098 bp,contains 375 amino acids,isoelectric point of 4.88,and a molecular weight of 37887.21 KDa.From the homologous alignment of amino acid sequences,the sequence similarity of HsfB2 gene in RcHsf1 and Arabidopsis thaliana,grape and tomato was extremely high,and the sequence similarity between RcHsf16 and HsfA2 gene in Arabidopsis thaliana,grape and tomato was extremely high.5.The expression vectors of RcHsf1 and RcHsf16 genes were constructed,we transferred RcHsf16 into Arabidopsis thaliana,RcHsf1 and RcHsf16 into the seasonal'Samantha'somatic embryos to analyze the function of the two genes.The results showed that compared with WT Arabidopsis thaliana,RcHsf16 verexpressing Arabidopsis thaliana showed better survival under high temperature stress.Under high temperature stress,the SOD activity of RcHsf16 overexpressing lines was higher than that of wild strains,the relative conductivity is significantly lower than that of wild strains,and the proline content accumulates and the soluble protein content increased more than wild strains.It also indicated that overexpression of RcHsf16 Arabidopsis increased the heat tolerance of transgenic plants to some extent.It has been initially confirmed that the RcHsf1 and RcHsf16 genes have been successfully transferred into the 'Samantha' Rose cells,and get resistant buds,and the function of the gene needs to be verified in the next step.