Cloning And Expression Analysis Of14-3-3Protein Genes In Cunninghamia Lanceolata (Lamb.) Hook.

The14-3-3protein family is a kind of acid and soluble protein,which was first discovered inthe study of classification of bovine brain tissue protein purification by Moore and Perez in1967.The14-3-3family is widely expressed,highly conserved,various function and which actsmainly as homodimers or heterodimers.14-3-3proteins encoded by different genes in differentspecies, but its amino acid sequence is highly homologous.14-3-3protein extensively involved invarious physiologic processes,such as the metabolism of plants、cell signal transduction pathway、gene transcription、resist disease and abiotic stress and the growth and develop of plant.So thereare widely attention and many research about it,but it has not been reported in the Chinesefir(Cunninghamia lanceolata).This study combine the method of designing degenerate primer andrapid amplification of eDNA ends to get the full length of14-3-3and check the accuracy of theresults by RT-PCR. Through the bioinformatics analysis of four14-3-3genes and gene expressionanalysis in order to explore Chinese fir the14-3-3gene’s function,and lay the base for exploreChinese fir functional genes for improve it on resistance to stress.The main results of this studyare as follows:1.In this study,four clones of Chinese fir14-3-3gene were gained by designing degenerateprimer from the14-3-3gene sequences of homologous species.Then,the5’end and3’ end weregained by using the RACE technology.Four Chinese fir14-3-3gene clones were obtained andverified.The results provide the base for the further functional analysis.2.We predicted and analyzed the four Chinese fir14-3-3genes by a variety of bioinformaticsanalysis softwares.The results showed that four14-3-3proteins are all acidic, the hydrophilic ofunstable protein and their molecular weight are close expect the14-3-3-3.Secondary structureprediction results showed that alpha helix is the highest proportion in the structure of the fourproteins.The results of biology function prediction is that14-3-3-0and14-3-3-4are likely toprotein transcription function,while the14-3-3-1and14-3-3-3are likely to amino acidbiosynthesis.The results of subcellular localization are showed that14-3-3-0,14-3-3-1and14-3-3-4are located in cytoplasm,but the14-3-3-3is located in extracellular.Transmembraneforecast reveal that14-3-3-3has a transmembrane helical area and predict it could be atransmembrane protein.The3-D prediction results showed that only the14-3-3-4protein is madeup of two monomers C type groove between forcipate structure space conformation.Amino acidhomology comparison analysis results showed that the four14-3-3proteins have the same conserved region with other species and different squences in the end.The molecular evolutiontree showed that14-3-3-0、14-3-3-1and14-3-3-3have close relationship with14-3-3proteinsfrom Picea glauca and Picea sitchensis while14-3-3-4have close relationship with14-3-3proteinfrom Solanum lycopersicum.All the results above lay a theoretical base for study the variety offunctions of the Chinese fir14-3-3family.3.Analyze the espression of the14-3-3genes in Chinese fir under the stress of drought byquantitative RT-PCR.The results of quantitative RT-PCR expression analysis showed that three14-3-3genes in Chinese fir have not been detected expect the14-3-3-3.The results provide thebase for the further study on explore the mechanism of Chinese fir14-3-3-3protein to droughtstress.