Isolation And Expression Of Genes Associated With Aroma Formation Genes In Process Of Fine Manipulation In Oolong Tea

Oolong tea is the main production of tea in Fujian Province,its production amounted to 197500 t in 2014,which accounting for 53%of the province's total tea.With a unique processing technology,it has heavy and pure fragrant and a good taste with mellow sweet.In a word,having high economic value and broad development prospects,it is well received by consumers all over the world.Tea aroma formation are closely related to terpene metabolism pathway,phenylpropanoid metabolism,lipid metabolism,amino acid metabolism,glycoside hydrolysis pathway and catechins metabolism.Fine manipulation of green tea leaves,the most important procedure while processing Camellia sinensis(L.)O.Kuntze cv.Tie-guanyin(Tie Guan Yin),is closely related with aroma formation.In this study,First,the research used Tie Guan Yin fresh leaves to construct Homogenization full-length cDNA library,and analysising EST sequence to find out the genes related to the formation of Tie Guan Yin aroma;Second,established Suppression subtractive hybridization cDNA library by utilizing Tie Guan Yin fresh leaves and "Sha qing qian" leaves,and Northern-blotting was used to screen the library to find out the aroma genes,then screened and separated genes closely related with aroma formation to predict function by EST sequencing and analysis;Finally,Detected aroma genes expression at different developmental stages,different parts of leaves and "Zuo qing" leaves of Tie Guan Yin by the fluorescence quantitative polymerase chain reaction(qPCR)technique,Through this can clarify the molecular mechanism in the fine manipulation of green tea leaves,and to provide a theoretical basis for aroma regulation,it has a important theoretical significance and application value for tea germplasm innovation,resistance breeding and development of high aroma tea products.The results of this study are as follows:1.Construct the Tie Guan Yin Homogenization full-length cDNA library and find out some important aroma formation genes of Tie Guan Yin.Total RNA was extracted from C.sinensis cv.Tie Guan Yin mature leaves,and two-step method of reverse transcription,inhibition LA-PCR and deal with DSN were used to construct the Tie Guan Yin Normalized Full-Length cDNA library,and find out some important aroma formation genes of oolong Tea.The results showed that the initial titer of the library was 1.97*105 cfu/mL,96.4%clones were recombinant,and the insertion fragment was 250-2 500 bp and the average size was about 1200 bp,the quality of library is well.A total of 211 clones were randomly selected from the library and sequenced,and 178 effective ESTs sequences were identified,which included 9 repeat and the redundancy rate is 5%,hence,in this work 169 ESTs were isolated.The functional annotation and classification of sequences were analyzed using Blast N research on the NCBI.The results showed that 107 ESTs were the functional genes(about 63%),36 ESTs were speculated function genes(about 21%),and 19 ESTs were the function unknown genes(about 11%),while 7 ESTs were known chromosomal location(about 4%).Fortunately,5 genes which related to Oolong tea aroma formation were found.They were NDR,IDI,NMT,HMGCL and SSL,Among of these genes,IDI,NDR and SSL are involved in the terpenes metabolic pathway,HMGCL is participated in lipid metabolism pathway,and NMT facilitates the benzene propane metabolism,and transcription factors such as bHLH,WRKY,MYB were obtained.2.Construct DSN mediated Suppression subtractive hybridization cDNA library,and some differentially expressed genes related aroma were obtained.Total RNA was extracted from C.sinensis cv.Tie-guanyin mature fresh leaves and "Sha qing qian" leaves,and two-step reverse transcription,ds cDNA amplification,ds cDNA hybridization,DSN treatment and differential cDNA amplification and other steps were used to construct the Tie Guan Yin SSH library.The results showed that the titer of primary library was about 4.3*104 cfu/mL,98%clones were recombinant,and the insertion fragment was 250-2500 bp and the average size was about 1800 bp,the quality of library is well.A total of 260 clones in the library were randomly selected to screening by reverse Northern-Blotting technology,a total of 62 positive clones were gotten,and the positive rate was 23.8%.Then sequenced and 60 effective ESTs sequences were identified,which included 2 repeat and the redundancy rate is 3.22%;The Blast N analysising showed that,34 ESTs were the functional genes,10 ESTs were speculated function genes,and 12 ESTs were the function unknown genes.Fortunately,4 genes which related to Oolong tea aroma formation were found.They were G-10-H?SHT?SAMT and XPT,which involved in the terpenes metabolic pathway,Moreover,the experiment obtained 14 ESTs related to stress resistance,and ASIL,ethylene-responsive element and other transcription factors.3.Bioinformatics analysis of 9 aroma genes(CsIDI,NDR,G-10-H,SAMT,XPT,SHT,SSL,HMGCL and NMT):The positive clones were sequenced and showed that the full length cDNA of CsIDI(1200 bp)contains a 717 bp open reading frame(ORF),encoding a protein of 238 amino acid,containing conserved domain of Idi and IPP isomerase locus;the full length cDNA of NDR(1075 bp)contains a 903 bp open reading frame(ORF),encoding a protein of 300 amino acid,containing conserved domain of carb-red-PTCR-like and fabG;the full length cDNA of G-10-H(1713 bp)contains a 1494 bp open reading frame(ORF),encoding a protein of 497 amino acid,containing conserved domain of P450-rel-GT-act,P450-cycloAA-1CypX,PLN02738 and MRS6;the full length cDNA of SAMT(1019 bp)contains a 744 bp open reading frame(ORF),encoding a protein of 247 amino acid,containing conserved domain of methyltransf and PLN02668;the full length cDNA of XPT(1568 bp)contains a 1320 bp open reading frame(ORF),encoding a protein of 439 amino acid,containing conserved domain of Eam and TPT;the full length cDNA of SHT(1532 bp)contains a 1365 bp open reading frame(ORF),encoding a protein of 454 amino acid,containing conserved domain of Condensation and BAHD superfamily;the full length cDNA of SSL(1395 bp)contains a 1137 bp open reading frame(ORF),encoding a protein of 378 amino acid,containing conserved domain of NHL and YvrE;the full length cDNA of HMGCL(1553 bp)contains a 993 bp open reading frame(ORF),encoding a protein of 330 amino acid,containing conserved domain of DRE-TIM-HMGL;the full length cDNA of NMT(1964 bp)contains a 1569 bp open reading frame(ORF),encoding a protein of 522 amino acid,containing conserved domain of rRNA-processing and nop2p.The amino acid sequence of the CsIDI share more than 94%homolog with Vitis vinifera,Astragalus membranaceus,Canarium album and Sesamum indicum;The amino acid sequence of the NDR share 68%-70%homolog with coffee,Lycopersicon esculentum and Populu;The amino acid sequence of the G-10-H share 65%-68%homolog with Cinchona ledgeriana,Ophiorrhiza mungos and Amsonia elliptica;The amino acid sequence of the SAMT share 99%homolog with Camellia sinensis,more than 50%with Theobroma cacao,Populu and Citrussinensis;The amino acid sequence of the XPT share 73%-76%homolog with Sesamum indicum,Nicotiana tabacum,and Populus euphratica;The amino acid sequence of the SHT share 54%-56%homolog with Nymphaea,Vitis vinifera and Pirus;The amino acid sequence of the SSL share 68%-73%homolog with Vitis vinifera,Ziziphus jujuba and Citrus reticulata Blanco;The amino acid sequence of the HMGCL share 77%-82%homolog with Aureobasidium pullulans,Aureobasidium pullulans and Acetobacter suboxydans.The amino acid sequence of the NMT share 70%-82%homolog with Vitis vinifera,Ziziphus jujube and Sesamum indicum.Generally speaking,IDI,NDR,G-10-H,SAMT,XPT,SHT and SSL belongs to MEP pathway,HMGCL belongs to lipid metabolism,NMT belongs to Benzene propane metabolism.4.To find out the Tieguanyin aroma related genes(CsIDI,NDR,G-10-H,SAMT,XPT,SHT,SSL,HMGCL and NMT)expression at different developmental stages,different parts of tea leaves and different stages durning the process of fine manipulation by using fluorescent quantitative PCR technology.Detection 9 genes dynamic changes in different developmental stages,different parts of leaves and "Zuo qing" different stages,GAPDH as a reference gene,the results show that,in terpenoid metabolic pathways CsIDI,NDR,G-10-H,SAMT,XPT,SHT and SSL expression along with the development of Zuo qing process were up-regulated.In the different parts of leaves,the expression of third and fourth leaves is higher than the first and second leaf,which is consistent to Tie Guan Yin picking standard.The pathways involved in lipid metabolism pathway of HMGCL in Tie Guan Yin Zuoqing process,the expression of this gene was up-regulated,and a larger increase in the rate;The expression of HMGCL in the developmental process of the propulsion and different parts of leaves downward,the expression was up-regulated,In agreement with picking Tie Guan Yin needs to be a certain maturity consistent.NMT involved in the phenylpropanoid metabolic pathway,the expression quantity in Tie Guan Yin Zuo qing process of dynamic expression were up-regulated and increased greatly,the development process and in different parts of the leaves dynamic expression with the development and maturity increased were increasing.In general,Tie Guan Yin leaves with a certain maturity have higher gene expression,which consistent with the study of picking standards which required certain maturity.