Identification And Analysis Of Acyltransferase Anthocyanins To Coumarin In Camellia Sinensis

Anthocyanins,which are naturally water-soluble pigments,have received increasing attention due to their extensive antioxidant activities,anti-cancer and cardiovascular disease prevention properties.’Zijuan’ is a representative purple tea variety,rich in anthocyanins.In recent years,with ’Zijuan’ as the research object,many researches on anthocyanin synthesis mechanism have been carried out.With the deepening of the researches,the basic biosynthetic pathway of anthocyanin has almost been clarified,but the dynamic change of the chemical structure of anthocyanin is still not completely clear.So far,four anthocyanins,namely 3-Ogalactoside,3-O-galactoside,3-O-(6-O-P-coumaryl)galactoside and 3-O-(6-O-P-coumaryl)galactoside of Delphinium,have been identified in ’Purple Run’.In a word,the enzyme responsible for the glycosylation of anthocyanins has been characterized.However,the enzyme responsible for acylation modification of anthocyanin has not been reported.CA3Ga6 CT in Arabidopsis Thaliana(coumaroyl-Co A: anthocyanidin 3-O-galactoside-6 "-O-coumaroyl transferase)is suggested to be the key enzyme that may acylate anthocyanidin 3-o-galactoside-6"-o-coumaroyl transferase on the 6 "of glycoside.In this study,we cloned the candidate gene CA3Ga6 CT from tea tree and analyzed its correlation with anthocyanin accumulation of’Zijuan’ and ’Ziyan’ at the transcription level.We constructed plant overexpression vectors and prokaryotic expression vectors,respectively,to clarify its subcellular localization and biological function through agrobacteria-mediated transient transformation of tobacco and Escherichia coli heterologous expression.The main research results are as follows:1.Using the BAHD domain HMM of Arabidopsis CA3Ga6 CT as probe,five CA3Ga6 CT candidate genes were screened from the tea tree genome and their ORFs were cloned.The ORF length of TEA014567.1 is 1371 bp,encoding 457 amino acids,molecular weight is 50.44 k Da,isoelectric point is 6.56.The ORF length of TEA005546.1 is 1344 bp,encoding 448 amino acids,molecular weight is 50.27 k Da and isoelectric point is 6.58.The ORF length of TEA021506.1 is 1365 bp encoding 455 amino acids,the molecular weight is 50.81 k Da,and the isoelectric point is 6.33.The ORF length of CSS000928.1 is 1386 bp,encoding 462 amino acids,molecular weight is 51.42 k Da and isoelectric point is 6.95.The ORF length of CSS0015285.1 is 1314 bp,encoding 438 amino acids,molecular weight is 48.94,isoelectric point is 6.27.All the five genes had two BAHD family conserved domains,HXXXD and DFGWG,and all had YFGNC motifs except CSS0015285.1.2.The content of acylated anthocyanins in different leaf positions of ’Zijuan’ and ’Ziyan’was determined,and its correlation with the expression levels of five genes was analyzed.The results showed that CSS0015285.1 and CSS000928.1 had similar expression patterns with the accumulation of acylated anthocyanins in ’Zijuan’,with the highest accumulation in leaves,followed by buds,and the lowest in stems.TEA014567.1 and TEA021506.1 showed similar expression patterns with the accumulation of two acylated anthocyanins in ’Purple Yan’.It is inferred that CSS0015285.1,CSS000928.1,TEA014567.1 and TEA021506.1 may have the function of acylation of anthocyanin-3-O-galactoside.3.Subcellular localization showed that all five genes were located in cytoplasm and nucleus.The experimental results were slightly different from the previous prediction,which was predicted by bioinformatics to be in the cytoplasm.However,according to the experimental results,CA3Ga6 CTs was indeed expressed in the cytoplasm(hyphen in the cell cavity is a typical structure of the cytoplasm),but it was also expressed in the nucleus.This means that tea tree CA3Ga6 CTs may be versatile and able to function in different subcellular locations.4.The prokaryotic expression vector was constructed,and the recombinant protein was induced by the four genes except CSS000928.1 under the conditions of 16℃,0.4 m M IPTG and 20 h,and the recombinant protein with high purity was purified.The acylation of anthocyanin galactoside to coumarin could not be catalyzed by in vitro acylation experiment and apple injection.It indicates that the recombinant protein has no acylation activity.In-depth understanding of the catalytic mechanism of tea tree anthocyanin acyltransferase is of great significance to reveal the metabolic pathway and regulation mechanism of tea anthocyanin,and provide theoretical basis and practical guidance for the development of tea high-quality and high value-added products.