Cloning And Functional Analysis Of RcHSP17.8 Gene Encoding A Small Heat Shock Protein In Rosa Chinensis

Rosa chinensis,which belongs to Rosaceae,is an important continuously blossomed ornamental plant.It plays a key role in the garden ornaments for its various genotypes and colors.So more and more attentions were paid on the researches of Rosa chinensis.With the increasing global greenhouse effect,high temperature has become the main restrictive factor for plant growth and development.Therefore,the research on how to enhance the abiotic stress tolerance such as thermotolerance of Rosa chinensis has large significance.Molecular biology researches of Rosa chinensis are based on high quality extracted DNA and RNA,which is affected by high abundant tannin,polysaccharides,and phenols that contained in the plant body of Rosa chinensis.In this study,we extracted high quality DNA from Rosa chinensis through improved CTAB method,and verified that RNAout kit is suitable for extracting RNA from Rosa chinensis.Meanwhile,we had also constructed an effective rapidly subculture system for it.In this work,we selected two Rosa chinensis varieties,Sohloss Mannieim(SM) and Kordes'Perfecta(KP),which shows different tolerance to high temperature,SM as resistant and Kpas sensitive one.These two varieties of Rosa chinensis were placed at 38℃for 3 hours,and two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) was used to analyze the protein expression pattern of these two varieties.Several spots which only appeared on the SM gel map were found out and the massspectrum and BLAST analysis implied that one of these spots might be a peptide of a small heat shock protein 17.8(sHSP 17.8).Open reading frame(ORF) of the HSP17.8 gene was attained by homology cloning, inverse-PCR and 3'-RACE.The ORF is a 465bp fragment,and encoding 154 amino acids,and was designated as RcHSP17.8(GeneBank accession number:EF053229). The deduced amino acid sequence was aligned with other representative sHSPs and demonstrated significant homology to sHSPs of other plants.Phylogenetic analysis revealed RcHSP17.8 to be a cytosolic class I sHSP.RT-PCR revealed that RcHSP17.8 is a high temperature induced expressing gene, and is rapidly induced expressing after 5min heat stress treatment(38℃),and after 5h, the mRNA level began to decline.RcHSP17.8 is more strongly expressed in flowers than that in other organs of Rosa chinensis under heat stress treatment.In addition, NaCl,PEG4000,sucrose and H₂O₂ also induce the expression of RcHSP17.8.Recombinant RcHSP17.8 was overexpressed in Escherichia coli(BL21) using pET32a,and also in yeast(SMD 1168) by the vector of pPIC3.5K to study its possible function under stress conditions.The recombinant E.coli and yeast cells that contained RcHSP17.8 showed improved viability under different stress conditions, such as high and low temperature,high salt concentration,heavy metal and oxidative stress,as compared with control cultures.Transgenic Arabidopsis thaliana that constitutively expressed RcHSP17.8 exhibited increased tolerance to thermal,salt and osmotic stress.These results suggest that expression of RcHSP17.8 enhances the thermotolerance and other abiotic stress resistance in both prokaryotes and eukaryotes. Besides,expressing of stress-correlative genes in Arabidopsis thaliana such as HSP101,Osmotin,BADH,P5CS and APX are parallel with the expressing of RcHSP 17.8.We also isolated promoter sequences of RcHSP17.8 from two varieties,SM and KP using inverse-PCR.Sequence alignment revealed that special elements existed in the promoter of SM,maybe it is related to regulate the expression of target gene.Some cis-regulate elements,such as HSE,LTR,ABA,MBS existed in both of promoter which are correlative with temperature and ABA stress regulation.