Abstract
BackgroundHeat shock proteins (HSPs) are found extensively in Eukaryotes and are involved in stress tolerance. However, their functions in herbaceous peony (Paeonia lactiflora Pall.) under high temperature stress are poorly characterized.ResultsIn this study, the genomic sequence of P. lactiflora HSP70, designated PlHSP70, was isolated. Its full-length was 3635 bp, and it contained a large 1440-bp intron. The encoded protein with a molecular weight of 71 kDa was localized in the cytoplasm of the cell. PlHSP70 transcription was detected in P. lactiflora and increased with the treatment of high temperature stress. The constitutive overexpression of PlHSP70 in Arabidopsis thaliana obviously conferred tolerance to high temperature stress by affecting different physiological and biochemical indices. Transgenic A. thaliana plants exhibited higher chlorophyll fluorescence values than the wild-type (WT) when exposed to high temperature stress. The accumulation of hydrogen peroxide (H2O2), superoxide anion free radical (O2·-) and relative electric conductivity (REC) were significantly lower in the transgenic A. thaliana plants compared to the WT. In addition, more intact cell membranes, chloroplasts and starch grains, and fewer plastoglobuli were found in the PlHSP70-overexpressing transgenic lines than in the WT.ConclusionsAll of these results indicated that PlHSP70 possessed the ability to improve the tolerance to high temperature in transgenic A. thaliana, which could provide a theoretical basis to improve high temperature tolerance of P. lactiflora by future genetic manipulation.
Highlights
Heat shock proteins (HSPs) are found extensively in Eukaryotes and are involved in stress tolerance
Cloning and sequence analysis of genomic DNA The genomic sequence of PlHSP70 was obtained using polymerase chain reaction (PCR) method based on its full-length Complementary DNA (cDNA) sequence (JN180465) and extracted DNA
The soluble fractions from the recombinant strains differed from those of the non-IPTG-induced pET-PlHSP70-sumo rose and empty pET-sumo rose vector, and the apparent molecular weight of the PlHSP70 protein plus the GST-Tag protein (16 kDa) was approximately 87 kDa, the molecular weight of the PlHSP70 protein was approximately 71 kDa, which was consistent with its putative molecular weight (Fig. 2)
Summary
Cloning and sequence analysis of genomic DNA The genomic sequence of PlHSP70 was obtained using PCR method based on its full-length cDNA sequence (JN180465) and extracted DNA. The DNA extracted from the leaves was used for identification using PCR and the results showed that one clear and bright band could be observed in the PlHSP70-overexpressing transgenic lines, while there was no specific band in WT (Fig. 5b). The subsequent high temperature treatment showed that the overexpression of PlHSP70 substantially enhanced the basal resistance to high temperature stress in transgenic lines compared to WT plants, and qRT-PCR analysis confirmed the significantly higher transcript levels of PlHSP70 in transgenic lines, suggesting that PlHSP70 might act as a molecular chaperone to confer plant high temperature tolerance. Our results revealed that Fv/Fm, Y (II), qN and ETR in PlHSP70-overexpressing transgenic lines were all significantly higher compared with WT, which suggested that photoinhibition of photosynthesis in A. thaliana occurred due to high temperature stress, and PlHSP70 played an important role in alleviating the photochemical damage caused by high temperature.
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