Cloning And Sequence Analysis Of Three Genes Related To The Synthesis Of Amino Acids In Camellia Sinensis

The3’ sequence of GS1-1gene, the full-length cDNA of GS1-2gene and argininedecarboxylase (ADC) gene were obtained by SMART RACE PCR method on the basis ofthe ESTs from young root cDNA library.After3’ sequence of GS1-1splicing with the known EST sequence, a cDNA sequencewith2906bp in length with an open reading frame (ORF) of2532bp, encoding843aminoacids with deduced molecular weight of93.553kDa and theoretical pI value of5.373wasobtained. Bioinformatics analysis showed that in this protein, α-helix structure andβ-pleated sheet structure accounted for41.52%,10.79%, respectively, and randon coil andother structures accounted for47.69%, which revealed that the most important structuralelement of the protein was randon coil structure，and the next was α-helix and the last wasβ-pleated sheet. GS1-1is a hydrophilic and unstable protein without any signal peptide andits transmembrane direction is from the inside to the outside. GS1-1is non-secreted proteinand it functions in cell nucleus. The analysis result of the genetic and molecular evolutionshowed that the GS homologue from Camellia sinensis has a close genetic relationshipwith Vitis.The full-length cDNA of GS1-2gene is1710bp in length with an open reading frame(ORF) of1071bp, encoding356amino acids with deduced molecular weight of39.34kDaand theoretical pI value of5.65. Bioinformatics analysis showed that in the secondarystructure of GS1-2, α-helix structure, β-pleated sheet structure accounted for26.29%,23.03%, respectively, and randon coil and other structures accounted for50.28%, whichrevealed that randon coil structure is the most important structural element of the protein，and α-helix and β-pleated sheet are scattered in the protein. GS1-2is a hydrophilic andunstable protein without any signal peptide, has no transmembrane domain. There are27phosphorylation sites within the polypeptide chain. It is a non-secreted protein and itfunctions in peroxisome. The analysis result of the genetic and molecular evolution showedthat the GS homologue from Camellia sinensis has a close genetic relationship with Vitis.The full-length cDNA of ADC is2988bp in length with an open reading frame (ORF)of2163bp, encoding720amino acids with deduced molecular weight of77.430kDa andtheoretical pI value of5.373. Bioinformatics analysis showed that ADC is a hydrophilicand unstable protein without any signal peptide, has no winded helix structure andtransmembrane domain. There are41phosphorylation sites within the polypeptide chain.ADC is non-secreted protein and it functions in chloroplast stroma. The analysis result of the genetic and molecular evolution revealed that ADC from Camellia sinensis has a closegenetic relationship with Prunus persica Batsch var. duplex Rehd and Malus domestica.A prokaryotic expression system for GS1-2was constructed. GS1-2gene was clonedinto plasmid vector pET32a and transformed into E.coli Rosetta strain. The recombinantwas reduced by IPTG with the concentration of1mM at37℃for4h, and a protein withmolecular weight of39.5kDa was obtained. The result is in correspondence with the initialprediction. Quantitative analysis through Image Acquisition and Analysis Softwarerevealed recombination protein of GS1-2accumulated up to47.1%of the total bacterialprotein.The obtaining of full-length cDNA of GS1-1, GS1-2and ADC gene would provide afoundation for further exploration of the function of GS and ADC in nitrogen metabolismand theanine biosynthesis.