The Preliminary Molecular Mechanism Of Special Catechins Metabolism In Camellia Ptilophylla Chang

Tea is a healthy beverage in the 21 st century,containing many biological active ingredients,such as tea polyphenols,caffeine,amino acids.The quality and health value of tea depends largely on the catechins whichare the main ingredients of polyphenols.Catechins in tea can be divided into epicatechins and nonepicatechins,and the content of catechins in general tea is dominated by epicatechin,especially the EGCG.Camellia ptilophylla chang is a special tea varieties founded in Guangdong province and exhibited high content in nonepicatechins,especiallyin GC G and C,showing a very special catechin metabolism characteristics.In order to clarify the molecular mechanism of special catechinmetabolism in Camellia ptilophylla chang,Camellia ptilophylla chang was selected as main research material and Yinghong 9 as control.High throughput sequencing was conducted on transcriptional profiling of tea plants followed by bioinformatic analysis.The genes involved in the synthesisof catesins including leucoanthocyantin reducase gene(LAR)and anthocyanin reductase gene(ANR)were isolated byc DNA library screening and RT-PCR technology.Sequence analysis and comparison was conducted on the isolated genes,and gene expressionlevel in these selected teawas determined by Quantitative real-time PCR.The gene prokaryotic expression system was constructed by DNArecombinant technology.In order to elucidate the protein expression of the target genes in tea,the specific peptides from LAR and AN Rwere selected for the preparationspecific antibodies,the main results are as follows:(1)The analysis of catechins content in selected tea plantsThe content of catechins in teawasdetectedthrough HPLC,the results showed thatCamellia ptilophylla changrich in nonepicatechins,especially GCGaccounts for the highest concentration and reached 5.24%,followed by Cwhich accounts for 1.8%.On the contrary,Yinghong 9given priority in epicatechins content,and EGCG,one of the mainepicatechins,accounts forthe highest content and reached1.68%.(2)The transcriptome sequencing and data analysisByhigh throughput RN A-sequencingcombined with bioinformatics analysis on thesequenced data,79958 and 87359 Unigenes obtainedfrom Camellia ptilophylla changand Yinghong 9respectively,and23378 and 22510 Unigenes were found up-regulated and down-regulatedin Camellia ptilophylla chang compared with Yinghong 9respectively.Functional annotation coupled with expression analysisof differential genesshowed that there were 513 differentialgenes involved in the synthesis of tea catechin.LAR and ANR,the two key genes modulated the synthesis of catechin,owned different expressing profiling,but all exhibiting lowerexpression level inCamellia ptilophylla changthan in Yinghong 9.(3)The cloning of the LAR and ANR genesUsing cDN A library screening technology and RT-PCR technique,LAR and ANR genes were isolated from Camellia ptilophylla chang(MY)and Yinghong 9(YH),and named as YH-LAR(1182 bp),MY-LAR(1112 bp),YH-ANR(1339 bp),MY-AN R(1014 bp)respectively.Sequence comparison showed the cloned gene not only ANRbut alsoLAR fromthe two tea plants shared same coding region and had identical deduced amino acids.LAR's coding regions include 1029 bp and encode 342 deduced amino acids,and ANR's coding regions include 1014 bp and encodededuced 337 amino acids.YH-LAR and MY-LARshared more than 95% homology to tea LARlanded inNC BI.YH-ANR and MY-AN Rexhibited high homology to tea CsANRlanded inNCBIwhich divided into CsANR1 and CsANR2,and our cloned two AN R genes involvedinto CsANR2 onthe basis of their sharinghomologymore than 97%.(4)The Q uantitative real-time PCR analysis of the LAR and AN R genesAccording to the results from q RT-PCR,the expression of the LAR and AN R genes in Yinghong 9tea leaves is higher than that ofCamellia ptilophylla chang.The relative expression level of the LAR gene in Yinghong 9 is surprisingly 16.7 times than that of Camellia ptilophylla chang,and the ANR gene expression levelwas 3.6 times greater in Yinghong 9 than that in Camellia ptilophylla chang.(5)The prokaryotic expression and the preparation of polyclonal antibodies of the LAR and ANR genesBy the use of recombinant DNA technology,the two cloned genes recombined into prokaryotic expression vectorp ET-32a(+)and gained p ET-32a-LAR and p ET-32a-ANR.By careful analyzing on deduced protein secondary structure,antigenicity,polarity and chargeofLAR and ANR genes,the high specificity short peptidescorresponding to LAR and ANRwereselected respectivelyfor the following preparation of polyclonal antibodies.