Preliminary Studies On Gene Function Of Cpglp In Chimonanthus Praecox (L.) Link

Germin-like proteins(GLPs)encoded by multigene family are glycoproteins and similar to Germins in structure.They are frequently retained in the extracellular matrix by ionic bonds.Most are very stable oligomers.GLPs exist in plants' all organs,tissues and developmental stages,play important roles in plants' growth and development.Research discoveries GLPs exit as enzymes, structural proteins,or receptors in plants'cells.in this paper a full-length gene edcoding germin-like protein from Chimonanthus Praecox's mixed cDNA library which is constructed by our laboratory, is isolated,named Cpglp(GeneBank accession number:EU116342).We have analyzed the cDNA sequence by bioinformatics and construct a expression vector,then transform tobaccoes,at last, identify the functions of exogenous gene.The research contents and results are as follows:1.Bioinformatics analysis of Cpglp and its encoding proteinAnalyze the cloning gene sequence by software DNAStar,DNAMAN and so on.The analysis shows that the cDNA is composed of 922 nucleotides,including 645 nucleotides ORF and encoding 214 amino acids.The protein' theoretical molecular weight is 22.3kDa,isoelectric point is 6.08.Amino acid sequence alignments is carried out on NCBI website.The results showed that there are 46 proteins which have high identities with CpGLP,GLP of Vitis vinifera is the first,identity is 79%.2.Construction of expression vectorCpglp gene is in the cDNA library cloning vector pTriplEx2,activate bacterial liquid respectively including pTriplEx2 and pMD-18TM and extract plasmid,then digest plasmid by restriction endonuclease SfiI.Recycle target gene and then link to cloning vector pMD-18TM.The recombinant plasmid pMD-18TM-Cpglp is successfully constructed and confirmed by restriction endonuclease digestion and PCR.Digest pMD-18TM-Cpglp by double restriction endonuclease XbaI,SmaI,then recycle the target gene and link to expression vector pC2301.The identification of PCR and enzyme cutting show that the construction of the recombinant pC2301-Cpglp plasmid could be confirmed. 3.Obtaining Km-resistant plants by agrobacterium tumefaciens-mediated transformationThe target gene was introduced into tobacco genome by leaf disc transformation,and obtain 28 Kin-resistant regenerating tobacco plants.4.Molecular biological identification of transgenic tobacco plants.Results of GUS staining analysis show that 18 of those plants' rootes which are Km-resistant plants are stained,this confirm the foreign gene is integrated into the tobacco genome.After verified by GUS staining,extract the positive plants'DNA,PCR amplification show that the positive plants have a specific band that is coincident with the expected size,but the control is not.Extract total RNA of transgenic tobacco plants,and then detect the expression of Cpglp,the results show that there is a specific band that is coincident with the expected size,but the control can't this confirmed that the target gene can be translated to RNA.5.Sensitivity expriment of transgenic tobacco plants to auxin.GLPs maybe exist in extracellular matrix as a auxin receptor.Multiple alignments are performed with NCBI proteins database,the results show that CpGLP have a high identity with GLP of prunus persica which have auxin-binding ability,amino acid identity is 73%.Inoculate transgenic tobacco leaves and the control approximate on differentiation medium supplemented with 1mg/L 6-BA and 50mg/L Km(the control is not),and different NAA concentrations,there are approximte 30 explants treated with every different concentration of NAA.After one month,record the number of adventitious buds differentiated by 16 explants treated by different concentration of NAA,and then compare the number of adventitious buds differentiated by transgenic tobacco explants and the control.The results show that the protein encoded by Cpglp don't have the function of binding auxin.6.Salt stress expriment of heat-shock transgenic tobacco plantsImmerse 6 transgenic tobacco plants and 6 control respectively into culture bottle,and place them in artificial climate box under 45℃for 5 minutes,tombacco plants are transferred into 250mmol/L NaCl solution.Compare the change of phenotype of the transgenic tobacco plants and the control.24hours later by salt stress treatment,discovery 2 transgenic tobacco plants wilt as well as the control,the phenomenon reveal that foreign gene are silent in the 2 transgenic tobacco plants;48 hours later by salt stress treatment,discovery 4 transgenic tobacco plants wilt slightly compared with the control.The result show that the transgenic tobacco plants have a strong ability to adapt to salt stress after heat shock treatment.