Cloning And Functional Study Of HSP70 Gene In Herbaceous Peony (Paeonia Lactiflora Pall.)

Herbaceous peony(Paeonia lactiflora Pall.)is a traditional flower in China.It is mostly distributed or cultivated in North China,Northeast China,Northwest China,and Southwest China,because it likes cool and dry environment.In recent years,with the widespread use of P.lactiflora in garden and the rise of the its high-grade cut flower market,Hangzhou of Zhejiang Province,Yangzhou of Jiangsu Province,Shanghai and other "Yangtze River Delta" regions have transplanted peony from Heze of Shandong Province,Luoyang of Henan Province and other major northern producing areas.However,due to the long duration of summer high temperature in the south of the Yangtze River in China,most of the excellent P.lactiflora cultivars showed poor growth and were unable to display their superior ornamental traits.Summer high temperature has become one of the main factors restricting the large-scale production of P.lactiflora cut flowers in southern China and its widespread use in landscape.So it is very important for cultivating P.lactiflora varieties with high temperature tolerance to find key genes regulating the high temperature tolerance of P.lactiflora.Therefore,this study cloned P.lactiflora HSP70 gene and performed bioinformatics analysis,and then clarified the expression characteristics of P.lactiflora HSP70 gene through its expression pattern,heterologous expression and subcellular localization.Moreover,the over-expression vector of P.lactiflora HSP70 gene was constructed,which was genetically transformed into Arabidopsis thaliana and the effects of high temperature stress on the transgenic plants were observed.Finally,the function of P.lactiflora HSP70 gene in regulating the high temperature tolerance of P.lactiflora was clarified,and the main results were as follows:(1)The full-length cDNA sequence of P.lactiflora HSP70 gene was cloned by RACE technology.The sequence was 2195 bp in length with a 5' non-coding region(70 bp),an initiation codon,a complete open reading frame(1953 bp),a stop codon,3' non-coding region(175 bp)and poly(A)tail(12 bp),encoded 650 amino acids.It had high homology with the reported nucleotide and amino acid sequences of HSP70 genes from other plants with accession number JN180465,which was named PlHSP70.(2)The DNA sequence of PlHSP70 gene was obtained by PCR amplification.The sequence was 3636 bp in length,including 2 exons and 1 intron,and the intron sequence conformed to the "GT-AG" splicing regularity,and the accession number was MF817498.Subsequently,the molecular weight of PIHSP70 protein was determined to be 71 kD by the expression of the recombinant plasmid in E.coli.And it was clarified that PlHSP70 gene was located in the cytoplasm by subcellular localization of PIHSP70-GFP fusion transient expression vector by transforming into rice protoplasts.(3)The expression pattern of PIHSP70 gene was detected by qRT-PCR.Under high temperature stress,the expression level of PIHSP70 gene gradually increased with the prolonging of stress time,which was consistent with the trends of REC,H2O2 and O2·-accumulation and MDA content in plants,but contrary to the relative water content in leaves.(4)An over-expression vector pCAMBIA1301-PlHSP70 was constructed when PIHSP70 gene was the target gene,which was transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thawing method and invaded Arabidopsis thaliana by Agrobacterium-mediated floral-dip method.The harvested T0 seeds were initially screened on MS medium containing antibiotics to give transformed T1 Arabidopsis thaliana.Meanwhile,PlHSP70-overexpressing transgenic plants were identified by GUS staining and PCR amplification.(5)Under high temperature stress,PlHSP70-overexpressing transgenic plants had higher temperature tolerance than that of wild-type Arabidopsis thaliana,which grew well,and leaves did not turn to yellow and shrunk.When compared with wild-type Arabidopsis thaliana,chlorophyll fluorescence parameters including Fv/Fm,Y(?),qN and ETR were all higher,relative water content of leaf was higher,whereas H2O2 and O2·-accumulation and REC was lower,and the expression levels of three protective enzyme genes including AtCo/ZnSOD,AtCAT and AtAPX were relatively higher in PlHSP70-overexpressing transgenic plants.Additionally,PlHSP70-overexpressing transgenic plants had more intact cellular structure with more chloroplasts and starch grains and less lipid globules compared with wild-type Arabidopsis thaliana.