Echanisms Of Volatile Terpenoid Biosynthesis In Tea Plants

In tea volatile compounds,monoterpenes and sesquiterpenes have great influence on aroma quality of finished tea.The aim of this study is to explore the biosynthesis of main monoterpenes and sesquiterpene aroma compounds in tea plants and its regulatory factors,so as to improve the aroma quality of tea.In this study,the terpene compounds in fresh leaves of six different tea varieties were analyzed and identified by solid phase microextraction?SPME?and gas chromatography mass spectrometry?GC-MS?.We have detected 45 terpene compounds.Linalool and geraniol are the main aroma components in the fresh leaves of tea,and the content of linalool in the"Yun Kang 10"is much higher than that of the other five species of Camellia,and the content of the flavanol is lower than that of the small leaf.This may be the key to the aroma difference of two kinds of tea varieties.In order to study the biosynthesis of nerolidol in tea,we cloned the nerolidol synthetase gene.The gDNA of CsNES was 4362 bp,with seven exons and six introns.It is a typical III type terpene synthetase gene.The CDs of CsNES was 1659 bp,encoding a protein of552 amino acids and 63.5 kDa,pI 5.46.Sequence analysis showed that the CsNES has a metal ion binding activity sites“DDXXD”and terpeng synthase activity sites“EDXXD”.Construction of prokaryotic expression vector,expressed the target protein in Escherichia coli,found that the expression of CsNES proteins as inclusion bodies.GPP or FPP can be used as the precursor of CsNES.A large number of linalool and a small amount of pinene are produced when GPP is used as the substrate.When FPP is the substrate,a large number of nerolidol and a small amount of?-farnesene and?-farnesene are generated,and CsNES shows a higher catalytic activity to FPP than GPP.PCAMBIA2300-CoxIV-CsNES plasmid was constructed to located CsNES into mitochondria and obtain transgenic Arabidopsis.In the over expression Arabidopsis,a higher level of?E?-nerlidol can be detected,but it can not be detected in the control of wild Arabidopsis thaliana.It is indicated that CsNES has the function of?E?-nerlidol in the plant.Subcellular localization indicated that CsNES was located in cytoplasm,indicatied that this enzyme is involved in the biosynthesis of?E?-nerlidol in cytoplasm.Moreover,CsNES has temporal and spatial expression and daily cycle characteristics,and the expression of genes is positively correlated with the generation of compounds.MeJA and GA₃ can induce the expression of CsNES,but the two peaks of induction were in different time periods.In order to study the biosynthesis of geraniol in tea,we cloned the Nudix hydrolase gene CsNUDX1.The CDs of CsNUDX1 gene was 663 bp and encoded 220 amino acids.The protein was predicted to be 24.3 KDa,and the pI was 5.54.The enzyme contains highly conserved Nudix box?Gx5Ex7REUxEExGU?,which is on the same branch with Arabidopsis AtNUDX1 and rose RhNUDX1.Expressed CsNUDIX1 in Escherichia coli was found unable to hydrolyze GPP to GP,but it can hydrolyze FPP to FP.Subcellular localization indicates that CsNUDX1 locates in plastids.Moreover,CsNUDX1 has temporal and spatial expression and daily cycle characteristics,and the expression of genes is positively correlated with the generation of compounds.Possible reasons for this correlation require further investigation.In order to study the biosynthesis of monoterpene and sesquiterpene in tea,we cloned the CsFPS gene of the tea,which was 1029 bp in length and encoded 342 amino acids.The protein was predicted to be 39.4 KDa,and the pI was 5.67.Construction of prokaryotic expression vector,expressed the target protein in Escherichia coli,found that the expression of CsFPS proteins as supernatant.CsFPS can catalyze IPP and GPP to generate FPP.Subcellular localization indicates that CsFPS is located in cytoplasm,indicating that this enzyme is involved in the biosynthesis of sesquiterpenes in cytoplasm.The pBI121-CsFPS plasmid was constructed and transgenic tobacco was obtained.Compared with the control wild type tobacco,there was no significant difference in the young period compaired with wild type.But in the mature stage,the growth of CsFPS overexpressed tobacco was smaller than wild type tobacco.In order to study the regulation factors of terpene synthetase gene in tea tree,we found that there were three miR167 precursors and mature sequences in the tea tree.The expression of miR167 is negatively correlated with the expression of target gene CsARF6and CsARF8,and has temporal and spatial expression and diurnal characteristics.MIM167can effectively inhibit the degradation of miR167 on its target genes CsARF6 and CsARF8.The transcriptional level of CsmiR167 in tea plant was significantly induced by MeJA,while the expression of CsARF6 and CsARF8 decreased significantly.In this study,the nerolidol synthetase gene,Nudix hydrolase gene and farnesyl pyrophosphate synthetase gene in tea were cloned.MIM167 was used to inhibit the expression of CsmiR167,in order to improve the volatile terpenoids of tea tree.It provides a theoretical basis for the study of the molecular mechanism of the temporal and spatial variation in the biosynthesis of terpenoid aroma substances in tea trees,and provides a theoretical basis for improving the aroma quality of the finished tea.It also has a reference to the improvement of the aroma quality of the related crops and the horticultural fruit tree.