Cloning And Functional Analysis Of PfHSP Genes Encoding Small Heat Shock Proteins In Primula Forrstii

Because of the number of varieties grown and its hold over the hearts and minds of the public, Primula is praised as one of the three great garden genera, only Rhododendron and Rosa can compare with it. With great wealth of species and high ornamental value, it is wildly sold as potted flowers; however, it could be also used in many other kinds of landscape decoration, such as parterre, rock garden, and bog and marsh garden. Approximately all species in this genus are distributed throughout the moister and cooler regions of the northern hemisphere where they are covered by snow during much of their resting period. Thus, high temperature becomes the primary barrier to cultivating them in warm areas. Therefore, it is interesting to research how to enhance the tolerance of plants to heat and other stresses. In our previous study, we found that Primula forrestii displayed the highest heat-resistance under heat stress compared with P. malacoides, P. obconica, P. veris, P. saxatilis and P. denticulatete. Through the suppression subtractive hybridization (SSH) method, three sHSP genes, PfHSP17.1, PfHSP17.2and PfHSP21.4, were highly expressed in the leaves of P. forrestii under high temperature treatment. In this study, we analyzed their expression patterns in P. forrestii under various abiotic stress treatments using RT-PCR. Then, the three genes were overexpressed in Arabidopsis thaliana and Escherichia coli to analyse their function.To elucidate the function of the three sHSP genes, recombinant plasmids expressing full-length PfHSP17.1, PJHSP17.2and PfHSP21.4were constructed. SDS-PAGE and Western blot analyses were used to confirm the expression of fusion proteins. The recombinant E. coli displayed enhanced viability under different stresses, such as high and low temperature, high salt and drought.PfHSP17.1was isolated from heat treated P. forrestii. Sequence alignments and phylogenetic analysis showed that PfHSP17.1belonged to sHSP cytosolic class I. Subcellular localization confirmed that PfHSP17.1localized in cytosol. The expression of PfHSP17.1was also triggered remarkably by salt, drought and oxidative stress conditions but was only slightly induced by abscisic acid (ABA). It was highly expressed in young leaves, old leaves, roots and stems, and a lesser extent in flowers under heat stress. Transgenic A. thaliana constitutively expressing PfHSP17.1displayed increased thermotolerance and higher resistance to salt and drought compared with wild-type plants.A novel small heat shock protein gene, PfHSP17.2, coding a protein of152amino acids was isolated from heat treated P. forrestii. Sequence alignments and phylogenetic analysis indicate that PfHSP17.2is a cytosolic class Ⅱ sHSP, which was further supported by PfHSP17.2-GFP fusion protein. PfHSP17.2was detected in developing and germinating seeds under normal conditions, and was highly expressed in leaves, roots, stems and flowers under heat stress. This gene was also strongly induced by cold, salt and oxidative treatments and to a lesser extent by drought and ABA stresses. Overexpression of PfHSP17.2in Arabidopsis enhanced tolerance to heat, cold and salt stresses.Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and HSP101, HSP70, APX and P5CS under heat stress was more pronounced in transgenic plants than in wild-type plants.These results highlight the important roles that PfHSP17.1, PfHSP17.2and PfHSP21.4play in diverse physiological and biochemical processes related to adverse conditions. This study paves the way for Primula sHSP genes future utilization in plant resistance breeding and facilitates the HSP research expanded to ornamental plants.