Genome-wide Identification,Evolutionary And Expression Analysis Of Terpenoid Synthase Gene Family In Camellia Sinensis

Tea(Camellia sinensis(L.)O.Kuntze)is a member of the genus Camellia in the Theaceae family,which belongs to the asterids of eudicots.Terpenoids are important components of tea aroma and one of the main components that determine the quality and flavor of tea.Terpenoid synthase(TPS)is a key enzyme in the synthesis of terpenoids,terpenoid synthase genes often replicate in tandem in the genome and not easily to be annotated.In this thesis,by integrating the genome and transcriptome data of the early version of Camellia sinensis var.sinensis cv.Shuchazao v1(CSS,Chinese type)and Camellia sinensis var.assamica cv.Yunkang10(CSA,Assam type),the TPS gene family of tea was systematically analyzed.Then the TPS genes were identified based on genome annotations of the seven tea plant genomes assembled based on threegeneration genome sequencing technology,including ‘Shuchazao v2’,‘Biyun’,DASZ,‘Longjing 43’,‘Tieguanyin’,‘Huangdan’ and‘Fudingdabai’.This thesis mainly elaborates the evolutionary history of the tea plant TPS genes,combined with gene expression data of different tissues and treatments,as well as the metabolism data of volatile substances in the leaves of ‘Fudingdabai’,to explore the function of the TPS genes.The main findings are as follows:1)Based on the annotated genome of tea plant and re-annotation on the genome,112 TPS sequences were identified in CSS type tea variety‘Shuchazao v1’(the first version),25 of them were newly annotated,and81 TPS sequences were identified in CSA type tea variety ‘Yunkang 10’,33 of them were newly annotated,which are more than most members of angiosperms,showing obvious expansion.The TPS gene family of tea can be divided into five subfamilies: a,b,c,e/f and g.In CSS type tea variety‘Shuchazao v1’,TPS-a and TPS-b have 32 sequences and 48 sequences respectively,which are the two largest subfamilies,and exceed the number of most plants in this thesis,which shows that tea plants are mainly expanded in TPS-a and TPS-b.2)In order to better understand the evolutionary history of the TPS genes of tea plant,combined with the TPS genes of other representative angiosperms,each subfamily of the TPS genes of angiosperms has been classified.Among them,TPS-a has one branch in angiosperms,TPS-b and TPS-g each have two branches in angiosperms,namely TPS-B1 and TPSB2,TPS-g.1 and TPS-g.2.TPS-a can be divided into 5 branches in eudicots,namely TPS-a.1-a.5;TPS-B1 has 4 branches in eudicots,namely TPS-b.1-b.4.TPS-B2,TPS-g.1 and TPS-g.2 have only one branch respectively in eudicots.TPS-c,TPS-e/f have only one branch respectively in angiosperms,and only one branch respectively in eudicots.3)The evaluation of the genome quality of ‘Yunkang 10’,‘Shuchazao v1’(the first version),‘Shuchazao v2’(the second version),‘Biyun’,DASZ,‘Longjing43’,‘Tieguanyin’,‘Huangdan’,and ‘Fudingdabai’ shows that the recently sequenced genome assembly is relatively well,but the annotation level is different.As TPS genes are generally clustered and difficult to annotate,the quality of genome annotation directly affects the annotation of TPS genes.Therefore,to study the TPS gene in detail cannot only rely on the annotation of the genome itself,but also manually annotate,that is,re-annotate.The number of TPS genes in the seven tea plant genomes mentioned above is quite different,for example,there are 94 in DASZ,while there are only 33 in ‘Longjing43’.According to the number of TPS genes in different tea plant genomes,it is estimated that there are about 100 TPS genes in each tea species.4)According to the classification of tea in angiosperms and eudicots,the TPS genes of tea plant can be divided into 12 branches,the TPS-a.2 and TPS-b.4 branches contain the most tea plant members.In CSS type tea variety ‘Shuchazao v1’,there are 23 and 33 respectively,and in CSA type tea variety ‘Yunkang 10’,there are 17 and 29 respectively.Among them,TPS-b.4 has more than 24 members in ‘Biyun’,DASZ,‘Tieguanyin’,and‘Fudingdabai’,and TPS-a.2 has 9,21,14,and 14 members respectively in the above-mentioned genomes.There are no other angiosperms inside the two branches other than tea,indicating that they are specifically expanding in the tea plant.Further analysis revealed that among the 12 branches of the tea plant TPS genes,except for TPS-c,which has only one copy in different varieties of tea plants,the other 11 branches have internal expansions,mainly tandem replication,and the number is quite different.The number of TPS genes in different tea plant genomes is quite different,in addition to the quality of annotations,it also shows that there are differences in the TPS genes of different varieties of tea plants.5)According to the transcriptome data of different tissues and treatments,the TPS gene expression of tea was analyzed preliminarily.TPS-a,TPS-b and TPS-g are highly expressed in apical buds,flowers and young leaves.TPS-g members are mainly expressed in flowers,while TPS-a and TPS-b members are mainly expressed in leaves,indicating that they should be the main members of terpenoid synthesis in tea.According to the protein encoded by the TPS-b branch gene of waterlily catalyzes sesquiterpene,it is speculated that the protein encoded by the TPS-b branch gene of tea may also catalyze the synthesis of sesquiterpene.CSS＿TPS107.g expressed in the treatments of low temperature,drought,salt and methyl jasmonate,indicating that it may has function in these treatments.According to coexpression analysis with terpene synthase genes,R2R3-MYB transcription factors Te MYB4 a.III-D,Te MYB21.III-A,Te MYB36 d.III-G,Te MYB64.IC,Te MYB65.II-C and Te MYB112.III-A may be involved in the regulation of terpenoids metabolic in tea plants.6)The volatile metabolites of ‘Fudingdabai’ were determined,and it was found that the content of heterocyclic compound was the highest,followed by terpenoids.Monoterpenes are the main components of terpenes,such as geraniol and linalool.According to gene expression,it is speculated that F1080315,which belongs to the TPS-b.4 branch,is the main terpene synthase gene that catalyzes the synthesis of monoterpenes.According to the co-expression network,more TPS genes were found to be specifically and highly expressed in flowers,indicating that TPS genes are more involved in flower development.In summary,the systematic identification and analysis of tea TPS gene family and gene expression provide a preliminary reference for future research on tea terpenoid gene function and breeding improvement in further studies.