Cloning And Functional Analysis On Rapid Alkalinization Factor Gene CpRALF From Chimonanthus Praecox(L.)

Rapid alkalinization factor (rapid alkalinization factor, RALF) belongs to a class of polypeptide hormone. At present, there are systemin, phytosulfokine (PSK), early nodulation 40 (ENOD40), S-locus cysteine-rich protein (PSCR), CLAVATA3 and rapid alkalinization factor (RALF) in plant. Systemin is the the first polypeptide signal molecule identified by Pearce in the research of system injury response.Then several other polypeptide hormones have been discovered. Rapid alkalinization factor is discovered in 2001 by Pearce G when he extracted and purified the tobacco system elements. Because the protein can cause a rapid alkalization of the medium of tobacco suspension cells, so it was named as the rapid alkalinization factor. Research shows that RALF has physiological functions of causing cell suspension culture medium alkalization, activation the intracellular MAPK, involving in the regulation of plant growth and development. And other study shows that external RALF proteins can inhibit the seed germination root elongation zone and make root hair loss and meristematic cell enlargement. RALF can regulate plant growth and development, however, whether RALF is involved in plant stress responses have not been reported.Chimonanthus praecox (L.) Link, a shrub originated from China, blossoms lordly in deep winter, possessing many advantages, such as resistance of coldness, drought, and secateur. Among them, chilling tolerance is its main characteristic. It is worth to research that Whether RALF is involved in the defense mechanism.We have cloned a RALF sequence from the cDNA library, with the Genebank accession number JQ217379. The main results are as follows:1 The cloning of CpRALF and characterization of the encoded proteinBased on a cDNA library constructed from Chimonanthus praecox flowers and EST analysis, a new gene was cloned by randomly cloning and sequencing, named as CpRALF. The CpRALF gene has an open reading frame (ORF) of 384 bp, encoding a putative polypeptide of 127 amino acid residues. Mature CpRALF protein contains conservative YIXY domain, YXRGC domain and four cysteine residues.2 Prokaryotic expressionAfter being inserted into an prokaryotic expression vector pET32a(+), CpRALF was transformed into and overexpressed in E.coli BL21(DE3). The result of SDS-PAGE indicated that the molecular weight of recombinant protein induced by IPTG was 27KDa which coincided with our expectation. The recombinant protein can make the alkalization of the medium,and can inhibit of root growth. The recombinant protein can make the tobacco suspension culture medium slight alkalization, and can inhibit the growth of Arabidopsis root.3 Eukaryotic expressionThe target CpRALF gene was linked with plant expression vector pCAMBIA2301g by restriction enzyme digestion, ligation, transformation, and then transformed into Agrobacterium tumefaciens LBA4404. The plant expression vector containing CpRALF was transformed into wild tobacco plants through Agrobacterium-mediated leaf disc method, nine strains of transgenic tobacco were obtained by means of resistance screening, GUS staining and PCR amplification test.4 Transcriptional Expression of CpRALFIt is showed that the expression level of CpRALF in Stems and flower is higher than that in stem and leaf by Real-time fluorescence quantitative PCR. And there is a lower expression level in Cotyledon and tender leaves. In the developmental stages of flower, the expression level of CpRALF in bud stage and wither period is more abundant than that in other periods. CpRALF was induced under various abiotic stresses such as high temperature, salinity and heavy metal. But the low temperature can inhibit the expression level of CpRALF. And the response of CpRALF is more quickly and strongly under stresses such as salinity and heavy metal.