Cloning And Function Analysis Of Key Genes Of Chlorogenic Acids Biosynthetic Pathway In Lonicera Japonica Thunb

Chlorogenic acid (CGA) is an important secondary metabolite during thedevelopment and growth of Lonicera japonica Thunb. Recently there has became ahot spot in the study of content of chlorogenic acid, synthesis mechanism andeffective development and utilization in the research field of biochemistry, molecularbiology and metabolic engineering. In order to effectively improve the chlorogenicacid production of Lonicera japonica Thunb, we investigated three key enzyme genesthat are important in biosynthesis of chlorogenic acid in Lonicera japonica Thunb.using research methods and tools from genomics, proteomics and bioinformatics. Theresearch results will help us understand the biosynthesis mechanism of chlorogenicacid, and also provide a certain reference for the application of gene engineering inthe field of metabolic engineering. The major results of this study were listed asfollows:(1) By RT-PCR and RACE method LjHCT, LjC3H1and LjCCoAOMT1that areclosely related to biosynthesis of chlorogenic acid in Lonicera japonica Thunb, wereisolated. LjHCT gene coding sequence (CDS) sequence is1293bp long, coding for430amino acids; LjC3H1gene is1533bp, coding510amino acids; LjCCoAOMT1gene is744bp, coding267amino acids. The phylogenic tree analysis showed that thesequences of LjHCT, LjC3H1, LjCCoAOMT1are most closely related to HCT gene inPlatycodon grandiflorus and Cynara cardunculus Linn., C3H1gene in coffee andTrifolium pratense L., and CCoAOMT1gene in Codonopsis lanceolata and Betulaluminifera., respectively. The three genes are more homologous to woody plants thanto herbs. Based on the prediction of transmembrane domain structure LjC3H1proteinhas one transmembrane structure domain, but the other two proteins have none.Subcellular localization analysis showed that LjHCT protein may play an importantrole in the chloroplasts or mitochondria, LjC3H1protein in the chloroplasts, andLjCCoAOMT1in the cytoplasm. Hydrophobic/hydrophilic analysis showed that allthree proteins are hydrophilic. The three proteins have alpha-helix and random coil bysecondary structure analysis. Amino acid conserved sequence analysis suggested thatLjHCT, LjC3H1, and LjCCoAOMT1proteins contain transferase conservativesequence, cytochrome P450conservative sequence, methyltransferase conservativesequence, respectively. (2) This study analyzed the CGA content in white flower, yellow flower, greenbud, white bud, young shoot, old leaf, young leaf and stem bark during flowerdevelopment in Lonicera japonica Thunb., and the results showed that the CGAcontent is the highest in green bud (2.95%of the dry weight), and lowest in the youngleaves and the old leaf, only0.20%and0.41%respectively. Fluorescence quantitativePCR analyses showed that LjHCT, LjC3H1and LjCCoAOMT1genes are widelyexpressed in different tissues of Lonicera japonica Thunb., but the expression levelsare different from each other. The expression of LjHCT gene is the highest in whiteflower, but lowest in white bud, and the relative expression of LjHCT gene in whiteflowers is400times higher than in white bud. The expression of LjC3H1genes is thehighest in yellow flower, but lowest in the old leaf, and the relative expression ofLjC3H1in yellow flower is20.5times higher than in the old leaf. The expression ofLjCCoAOMT1genes is the highest in green bud, but lowest in the white bud, and therelative expression of LjCCoAOMT1in green bud is439.1times higher in white bud,which showed that LjCCoAOMT1gene was expressed in different tissues, but therelative expression of the three different proteins differ greatly. The relativeexpression of LjCCoAOMT1gene reduces at earlier stage, and increase at later stage.(3) We expressed the coding proteins of LjHCT, LjC3H1and LjCCoAOMT1genes in E. coli BL21. By the Ni-chelating affinity chromatography methods, thethree kinds of soluble recombinant proteins of pCOLD-LjHCT,pCOLD-LjCCoAOMT1and pCOLD-LjC3H1were obtained. Using the coomassiebrilliant blue dyeing method, we found that the sizes of three fusion proteins(pCOLD-LjHCT, pCOLD-LjCCoAOMT1, pCOLD-LjC3H1) are92kDa,72kDa, and102kDa, respectively. The correlations between CGA content in different tissues andrelative gene expression of LjHCT, LjC3H1and LjCCoAOMT1were analyzed, and theresults showed that the chlorogenic acid content is highly correlated withLjCCoAOMT1gene expression, but this high correlation with chlorogenic acidcontent is not found in the other two genes. There was negative correlation betweenLjHCT and LjC3H1gene expression, but the correlation was not significant, whichmay be due to the reversible reaction that LjHCT catalyzes chlorogenic acid reactingto form caffeic acid. Using HPLC testing the change in substrate content, weidentified activity of the LjCCoAOMT1recombinant protein, and the results showedLjCCoAOMT1can catalyze caffeic acid to form ferulic acid.(4) We constructed three YFP fusion expression vectors, which are: YFP-LjHCT,YFP-LjC3H1and YFP-LjCCoAOMT1, using protoplast transformation method. The subcellular localization assays showed that LjHCT protein was located in differentorganelles，LjCCoAOMT1protein mainly in the cytomembrane, and LjC3H1proteinonly in the organelles, which are generally the same as the prediction frombioinformatics analysis. We constructed the pCAMBIA-1301::LjCCoAOMT1plantexpression vector, and transformed the vector in rice using the agrobacteriummediated transformation method. Compared to the wild type rice, the successfullytransgenic plants have no significant difference in plant height, spike length, leaflength, leaf width and grain weight, but does have significant difference in tillernumber and grain number per panicle.(5) We investigated the impact of environmental conditions on LjCCoAOMT1gene expression (light and gibberellic acid (GA3)) in Lonicera japonica Thunb.seedling, transgenic LjCCoAOMT1rice and wild type rice. The result showed that inthe dark conditions, Lonicera japonica Thunb. turned yellow, and transgenic riceturned yellow more seriously, but after being transferred to blue light environments,Lonicera japonica Thunb. turned green, and the LjCCoAOMT1transgenic rice turnedgreen more quickly than the wild type rice, which suggests that transgenic rice wasmore sensitive to light conditions than the wild type rice. Through the fluorescencequantitative PCR analysis, we found in that Lonicera japonica Thunb. the relativeexpression of LjCCoAOMT1increases under dark or blue light conditions, thetransgenic rice OsCCoAOMT1expression also increase more quickly than in naturallight. The result showed that gene expression patterns of Lonicera japonica Thunb.LjCCoAOMT1and rice OsCCoAOMT1are basically identical. Lonicera japonicaThunb., LjCCoAOMT1transgenic rice and wild type rice were treated with differentconcentration of GA3. We found that promotion effect of small dose of GA3onLonicera japonica Thunb. growth is not obvious, but GA3can effectively promoteboth transgenic LjCCoAOMT1rice and wild type rice growing, and the promotingeffect becomes greater with increasing concentration. However, the LjCCoAOMT1transgenic rice grew faster. The OsCCoAOMT1expression quantity increased alongwith the increasing of GA3in transgenic LjCCoAOMT1rice and wild type rice, butmore significantly in transgenic LjCCoAOMT1rice. When GA3concentrationincreased from200mg/L to400mg/L, OsCCoAOMT1gene expression in wild typerice did not increase obviously, but in transgenic LjCCoAOMT1rice the differencewas very obvious, which indicated that under high concentration the physiologicalactivity of GA3was weakened, and in LjCCoAOMT1transgenic rice, the GA3canstill perform their normal physiological function under high concentration. (6) This study successfully set up the rapid reproduction technology of Lonicerajaponica Thunb. The most appropriate explant is young stem, the most suitablemedium for callus induction is WPM+6-BA2.0mg/L+KT0.5mg/L+NAA1.0mg/L medium, the most suitable medium for cluster bud induction is WPM+6-BA2.0mg/L+KT2.0mg/L+NAA0.2mg/L medium, and the most suitable medium forcluster bud rooting induction is WPM+NAA0.1mg/L+activated carbon150mg/Lmedium. Using the young stem for explants, we successfully set up the callus cellsuspension culture technology system of Lonicera japonica Thunb., and successfullytransferred LjCCoAOMT1gene to suspended cells. In suspension cells of Lonicerajaponica Thunb., we used the fluorescent quantitative PCR method to detect therelative expression quantity of LjC3H1, LjCCoAOMT1and LjHCT. the result showedthat LjCCoAOMT1gene expression was strengthened in transgenic callus, the expressquantity being4.2times for wild type callus. The expression of LjC3H1gene in thetransformation LjCCoAOMT1callus strengthened too, but LjHCT gene in thetransformation LjCCoAOMT1callus was restrained, which may result from the factthat LjHCT enzyme is double active in the chlorogenic acid metabolic pathway.