Studies On Identification And Expression Of MiRNAs And Target Genes Involved In The Biosynthesis Of Catechins In Camellia Sinensis

Catechin,the important secondary metabolite and healthy component of tea,is essential for the quality of tea and has been widely used in food,medicine and daily life.MiRNA,a class of short non-protein-coding RNAs,plays important roles in many aspects of plant growth and development,including morphogenesis of organelles,signal transduction,response to hormones and environmental stresses and nutrition metabolism,as gene expression regulatory factors.Current studies about catechin synthesis regulation mainly focus on environmental or transcriptional factors,however,miRNAs' effects on plant growth and development,which has been developed to be a new angle for tea researches,has not been reported to be used in studying catechin synthesis regulation.This work focused on obtaining sequence information of tea miRNAs by high throughput sequencing and bioinformatics methods,which were further used to test their regulatory roles in catechin synthesis by RLM-RACE and degradome sequencing.Major results are described as follows:1.Computational identification of miRNAs of '1005' teaBased on the transcriptome library of '1005' tea and miRBase database,we identified 92 miNRAs in 80 families through the bioinformatic prediction method.To further confirm the existence of these miRNAs,we used qPCR to test the expression patterns of 31 miRNAs in the first,third and old leaves.These data indicated that these miRNAs predicated by bioinformatics method were truly existed,which in turn confirmed the reliability of this method.2.Identification of miRNAs of '1005' tea by high throughput sequencingWe identified the conserved and specific mirnas of tea using the combined tealeaves of 1005,by high throughput sequencing.We identified 73 conserved miRNAs and 42 novel miRNAs of '1005' tea by analyzing sequencing datas.3.Verification of miRNAs target genes of '1005' tea by degradome sequencingWe first constructed the degradome library using the combined leaves of tea strain 1005.We next identified 26 targets and 26 cleavage sites of miRNAs based on the miRNA sequences and transcriptome library of tea.The target genes mainly encoded proteins functional as transcription factors,resistance proteins and signal molecules and most the targets were homologous to that in Arabidopsis which indicating the conservation of these targets.4 Verification of cleavage sites of catechin biosynthesis pathway genesBased on the identified miRNAs and the catechin synthesis related genes in NCBI,we predicted multiple miRNAs related to catechin synthesis by psRNATarget.We next found 14 miRNAs could cleave 9 catechin synthesis pathway genes by RLM-RACE assay.9 catechin synthesis pathway genes were Cinnamate 4-hydroxylase(C4H),two Chalcone synthase(CHS),Chalcone isomerase(CHI),Flavanone 3-hydroxylase(F3H),Dihydroflavonol 4-reductase(DFR),Leucoanthocyanidin reductase(LAR)and two Anthocyanidin reductase(ANR).MiR5251 and miR7777-5P.1 target to a same gene,C4H.MiR7814 could target to CHS1 and CHS3 at two cleavage sites.MiR167a and miR529d targeted to CHI.MiR5240,miR3444b and miR4380a also cleaved the same gene DFR.MiR286 targeted to LAR.MiR156g-3p cleaved F3H at two sites.MiR5559-5p targeted to ANRI and ANR2.MiR2593e targeted to ANRI.MiR7717c-3p and mtr-miR5264 also targeted to ANR2.Besides,mtr-miR5264 could target to ANR2 at multiple sites.What's more,we also identified some other potential cleavage sites,which indicated that catechin synthesis may be regulated by some unknown miRNAs.5 Expression patterns of catechin synthesis genes and miRNAWe measured the catechin contents of tea leaves treated by different hormones,including ETH,2,4-D,IAA,ABA and 6-BA,and we found that all treatments induced similar trends of catechin contents except for some tiny differences.Generally,total catechin and ester catechin contents were similar after hormones treatments.They all decreased in the first and second leaves but increased in old leaves.However,the contents of non-galloylated catechins showed bigger fluctuation under different hormones treatments,for example,in the first leaves,their contents increased when treated with IAA,ABA or 6BA,but deceased under treatment of 2,4-D or ETH.The non-galloylated catechins contents significantly decreased in second leaves,but showed no clear differences in old leaves under all treatments.To figure out how the catechins contents were affected by exogenous hormones,we tested the expression levels of genes involved in catechin synthesis after hormone treatments.IAA treatment induced genes expressed variously in different tea leaves,for example,in first leaves,CHI and F35H were highly expressed than control while the expression levels of PAL,CHS1,F3H and ANS were lower than control;in the second leaves,4CL1 and CHI expression levels were significantly higher than control while ANS was lower,however,expression levels for most of the genes,including C4H,4CL2,CHS1,CHS2,CHS3,F3H,DFR,LAR,ANRI and ANR2,were decreased in old leaves except for PAL.These data demonstrated that most of the genes involved in flavonoid pathway were negatively regulated by exogenous 2,4-D and IAA,and further inhibit catechin synthesis.ETH treatment significantly reduced expression of PAL,CHS1,CHS2,CHS3,F3H,DFR and ANS but promoted F35H expression in the first leaves,however,this treatment increased the expression of most genes except for CHI in the second leaves.In the old leaves,ETH treatment significantly enhanced the expression of PAL,4CL1,CHI,ANS,but down-regualted the expression of CHS1,CHS2,CHS3,F3H,F35H,ANRI and ANR2.After treated with ABA,most of the genes' expressions were significantly promoted except for ANS in the first leaves.In the second leaves,the genes involved in phenylpropane and flavonoid pathways were all up-regulated.However,ABA treatment reduced most of the genes expression in old leaves except for ANS.Besides,we also tested how the catechin synthesis related genes were affected by 6-BA,the results showed that most of the genes' expression were promoted in both the first and second leaves except that F3H,ANS in the first leaves and CHI,F3H in the second leaves were not significantly affected.In the old leaves,expression of 4CL2,CHS1,CHS2,CHS3,CHI,F3H,DFR,ANR1 and ANR2 were down-regualted while other genes were not significantly affected.These data indicated that ABA and 6BA treatment may facilitate the synthesis of other products in the phenylpropane and flavonoid pathways,such as anthocyanidin,thus reduced the catechin synthesis and accumulation.We tested the expression of these miRNAs and their targets under treatments of different exogenous hormones.Expression of miR5251,miR7777-5p.1 and their common target C4H had negative correlation in old leaves but not in the first and second leaves.The results showed that the expression pattern of miR529d and miR167a were negatively correlated with CHI.miR286 and LAR have negative expression,indicating that miRNAs could negatively regulated gene expression at post-transcriptional level.It is possible that hormone treatments promote the synthesis of flavonoid and lignin and futher the C4H gene expression levels,which in turn,actives the negative feedback regulationg of miR5251 and miR7777-5p.1.miR5240,miR3444b and miR4380a,all targeting to DFR,had similar expression patterns under exogenous hormones treatments,indicating that the three miRNAs co-regulated the DFR expression.However,expression patterns of other miRNAs,including miR7814,miR156g-3p,miR5559-5p,miR5246,were not completely negatively correlated with their targets.We found that the expression of csn-miR2593e was opposite with its target ANR1 in the first and old leaves under hormone treatments,however,they were both up-regulated in the second leaves which suggesting different regulatory pathways maybe activated in leaves of different maturity or under different exogenous hormones treatments.6 Expressions and biological function of CsDHD/SDH of '1005' teaBased on the tea miRNAs we got,we identified 4 miRNAs that regulated CsDHD/SDH in tea.CsDHD/SDH2 was targeted by miR5180b,miR1510b-5p and miR24;CsDHD/SDH3 was targeted by miR868-5p.We also found that CsDHD/SDH2 and CsDHD/SDH3 had similar expression pattern under different hormone treatments and they were synergistic to each other.CsDHD/SDH1 had opposite expression pattern with the other two homologous and increased expression of CsDHD/SDH1 would lead to the reduction of the other two genes'expression.To further analyze the function of CsDHD/SDH,we made the interference constructs and transferred them to Nicotiana benthamiana and got the transgenic plants,T1 line.Real-time PCR analysis showed that NtDHD/SDH1 was down-regulated,but NtDHD/SDH2 was up-regulated in the transgenic plants.Besides,the flavonoid in the transgenic N.Benthamiana was reduced which indicated that DHD/SDH1 played essential roles in flavonoid biosynthesis.