Isolation Of Differentially Expressed Genes In Processing Of Withering And Expression Analyses Of Genes Encoding Key Enzymes In Pathways Of Catechins Metabolism In White Tea

White tea is mainly produces in Fujian.There is not panning and rolling during the processing,which is withering and rolling directly.Polyphenols,amino acids,chlorophyll,aromatic compounds,alkaloids is closely related to the quality of white tea.Using the fresh leaf and the withered leaf of C.sinensis cv.Fuding-dabaicha as the materials,to use DSN mediated suppression subtractive hybridization(SSH)cDNA library,to construct the fresh leaf-the withered leaf forward SSH cDNA library.Using reverse Northern Blotting to sift the clones includes the differential genes,and the analysis of clones are categorized by EST sequencing and analysis,the differential genes closely relates with quanlities formation to predict function by EST sequencing and analysis;Using fluorescence quantitative PCR technique to detect dynamic changes of expression quantity in gene involvement of quanlities formation in withering of White tea;The present experiment was conducted to investigate change of contents of tea polyphel and catechins components in withering process of white tea as well as dynamic expression of genes encoding key enzymes in their biosynthesis pathways,in order to provide theoretical basis for optimizing withering craft of white tea.It is of crucial theoretical value and practical significance:1、Some differential genes related to quanlity in process of White tea were obtained by construction and screening of the fresh leaf-the withered leaf forward-SSH cDNA library:optimum DSN concentration of treatment was 1/2 U,optimum cycling number of amplification of differential cDNAs was 20,The results showed that the titer of primary library was about 5.1*104 pfu/mL,93.52%clones were recombinant.The results of bacterial liquid PCR showed that every clone had inserts,and the size of the inserts was at the range of 650bp-2000bp.300 positive clones were screened by reverse Northen-Blotting technology,selecting 158 positive clones and the positive rate was 52.67%.The Blast N analysising showed that,112 ESTs were the functional genes,35 ESTs were speculated function genes,and 11 ESTs were the function unknown genes.3 genes which related to White tea quanlity formation were found.They were S-adenosylmethionine decarboxylase(SAMDC),Succinate dehydrogenase(SDH),Phospho-2-dehydro-3-deoxyheptonate aldolase(DAHPS).2、Analysis of 7 candidate reference genes of Expression stability values in the withering of White tea by GeNorm,NormFinder,BestKeeper the stability of β-Actin,GAPDH and RUBP gene was good.RUBP was verificated in candidate reference genes of Carnellia sinensis(L.)O.Ktze.The β-Actin gene could be used as the best candidate reference gene for different leaf positions;Due to the processing of tea,the external environmental factors had a greater impact,It was recommended to use GAPDH+RUBP gene combination of white tea processing process of the formation of related functional gene correction.3、Bioinformatics analysis and expression of 3 differential genes related to quatility(CsSAMDC、CsSDH、CsDAHPS):The positive clones were sequenced and showed that the full length cDNA of CsSAMDC(1549bp)contains a 1073bp open reading frame(ORF),encoding a protein of 337amino acid,share 87%homolog with Camellia sinensis,100%homolog with Cynodon dactylon、Triticum aestivum、Phaseolus vulgaris、Glycine max,result of real-time fluorescent quantitative PCR showed that CsSAMDC kept the increased and decreased at 0 hour to 16 hours and 24 hour to 52 hours,express the highest amount at 4 hours express the highest amount 4 times than 0 hour.The positive clones were sequenced and showed that the full length cDNA of CsSDH(1224bp)contains a 1150bp open reading frame(ORF),encoding a protein of 394amino acid,share 100%homolog with Litchi chinensis、Pinus massoniana、Vitis vinifera、Elaeis guineensis,result of real-time fluorescent quantitative PCR showed that CsSDH kept the increased a at 0 hour to 52 hours express the highest amount at 0 hour,express the highest amount 6 times than 52 hours.The positive clones were sequenced and showed that the full length cDNA of CsDAHPS(1935bp)contains a 1812bp open reading frame(ORF),encoding a protein of 560amino acid,share 100%homolog with Solanum lycopersicum、Ipomoea purpurea、Theobroma cacao,result of real-time fluorescent quantitative PCR showed that CsDAHPS kept the increased a at 0 hour to 24 hours,express the highest amount at 32 hours,express the highest amount 5 times than 52 hours.4、Using white tea at different stages of withering as materials,the contents of tea polyphel and catechins components in biosynthesis pathways of catechins were determined in leaves by Folin-phenol(Folin-Ciocalten)colorimetric method and HPLC.The expression quantities of key enzymes genes(PAL、C4H、CHS、CHI、F3 ’5 ’H,F3 ’H、F3H、DFR、LAR、ANS、ANR and UGGT)in biosynthesis pathways of catechins was determined by real-time quantitative PCR.The results showed that,tea polyphel content was decreased gradually.The contents of catechins components including epicatechin(EC),gallocatechin(GC),epigallocatechin(EGC)and epigallocatechin gallate(ECG)first decreased and then increased,reaching maximum at 32 h after withering.The results of real-time PCR showed that,the expression quantity of key genes PAL、C4H、CHI、F3H,F3’H,F3’5’H,DFR、LAR,ANR、UGGTin biosynthesis pathways of catechins increased at 32 h after withering,the expression quantity of CHS gene decreased as duration of withering increased.The expression quantity of ANS gene first decreased and then increased,reaching maximum at 16 h after withering.Furthermore,the expression quantities of genes encoding key enzymes played an important role in regulating biosynthesis pathways of catechins.In withering process of white tea,the expression of key genes PAL,C4H,F3H,F3’H,DFR,LAR,ANR in biosynthesis pathways of catechins is consistent with the accumulation law of catechins components(EC,GC,EGC),reaching Maximum at 32 h after withering.