| Strawberry is one of import crops in protected culture. There are some factors such as high/low temperature, salt and pathogens affecting the production and quality of strawberry. Although we can do somethings to control pathogens or to increase tolerance of strawberry to high/low temperature stresses, we have to pay more for this. It is important to increase tolerance of strawberry to above stresses using genetic engineer breeding. Plant calcium-dependent protein kinases (CDPKs) play vital roles as second calcium sensors during various developmental processes and responses to biotic and abiotic stresses. In the present study, we isolated and characterized a novel calcium-dependent protein kinase gene designated as FvCDPKl from a wild diploid strawberry accession Heilongjiang-3(Fragaria vesca L.). To further utilize FvCDPKl, we analysised the expression profile, the biochemistry characterization and subcelluar localization of this gene, also tested this gene functions in transgenic Arabidopsis and strawberry. The main results are as follows:1. The full-length cDNA of FvCDPKl has1825nucleotides, with an open reading frame of1653bp encoding a polypeptide of550amino acid residues. The deduced FvCDPKl protein contains the basic features of typical plant CDPKs:a catalytic kinase domain and a regulatory calmodulin-like domain containing four EF-hand calcium-binding motifs. Phylogenetic analysis showed that the FvCDPKl protein belongs to plant CDPKs IV subgroup.2. The cDNAs reversed transcripted from RNA of Heilongjiang-3leaves by treatmented such as low/high temperature, ABA, NaCl and SA were used for real-time qRT-PCR analyses. The expression of FvCDPKl can be induced by low/high temperature, especially low temperature, by which the FvCDPK1expression can be induced rapidly and reached at the highest level (almost five times to control) at one hours post treatment. This result indicated that FvCDPKl may be play import role of tolerance in strawberry to cold stress. In addition, real-time qRT-PCR analyses showed that FvCDPK1is predominantly expressed in meristems, but its transcript is also detected in other vegetative tissues, including roots, runners, young leaves, buds, and flowers. The expression of FvCDPKl in roots is much higher than the expression in runners, leaves, buds, and flowers. Furthermore, we found that the FvCDPKl expression in fruits was affected by developmental stages.3. Transient expression of FvCDPKl showed that FvCDPKl-GFP and GFP-FvCDPKl both targeted to the nucleus of onion epidermal cells by using particle bombardment. GST-FvCDPKl vector was constructed and the recombinant protein of GST-FvCDPK1expressed in Escherichia coli exhibited CDPK activity in vitro, and the activity depended on Ca2+.4. The p1301-FvCDPKl construct was introduced into Col-0by Agrobacterium-mediated flower dipping transformation. Ectopic over-expression of FvCDPKl in Arabidopsis T2plants showed enhancing tolerance to low temperature stress, and responsed to ABA.5. The optimum media of leaves regeneration and transformation of strawberry cv ’Confidante’ is MS media containing6-BA5.0mg/l and2,4-D0.1mg/1, and the frequency of adventitious bud induction was85%. The regeneration media containing25mg/1Hygromycin was better for transgenic plants selection. Transgenic strawberry plants were obtained from leaf disk transformation by PCR and RT-PCR analysis of FvCDPKl gene. The results showed that over-expression of FvCDPKl in strawberry maybe enhance tolerance to cold stress.6. According to the genome sequences of strawberry Fragaria vesca, promoter region of FvCDPKl was cloned from leaf genomic DNA of strawberry accession Heilongjiang-3(Fragaria vesca L.). The fragment was designated as PfvCDPKI, which was813bp in length. Transient expression of PfvCDPK1::GUS in the leaves of strawberry cv ’Zaohong’ tissue culture seedling showed that the activity of PfvCDPKl was induced by cold stress. PfvCDPKl was speculated as an cold induction promoter. |
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