Cloning,Functional Identification And Expression Analysis Of Strictosidine Synthase (CsSTR) Genes In Camellia Sinensis

The various alkaloids in tea plants play an important role,not only in plant stress resistance but also human medical treatment,especially strictosidine synthase acts as a throat position during the synthesis of alkaloids.so the study of strictosidine synthase in tea trees contributes to understand the metabolism of alkaloids and the development of tea tree quality,which has important theoretical and practical significance.In this study,four CsSTR genes were cloned from the national cloned variety“Shuchazao”.Expression of the cloned CsSTR genes was induced in E.coli and fall army worm,Spodopterafrugiperda.The catalytic function of CsSTR was characterized by using tryptamine and secologanin as substrates.At last,expression models of the CsSTR genes in different organs of tea plant and under different stresses treatment was carried out by employing q RT-PCR.The main results of this thesis are as follows:1.The CDS sequences of four CsSTR genes in tea trees were obtained by searching in the“Cloud Anti-No.10 Genome Database”.The full-length sequences of the four STR genes were successfully obtained by RACE technology,which were CsSTR1（1393 bp）and CsSTR2（1337 bp）,CsSTR3（1134 bp）,CsSTR4（1384 bp）,open reading frame were1208 bp,1173 bp,1134 bp,1128 bp,and the promoter sequence of CsSTR2 gene was obtained,and the sequence length was 2175 bp.2.Based on the cloning of the target gene,CsSTR2-pET30a（+）,CsSTR4-pET30a（+）,CsSTR2-pMALC2X,and CsSTR4-pMALC2X prokaryotic expression vectors were constructed and transformed into E.coli competent cell to induce protein expression;insect expression vector CsSTR2-pFast Bac1,CsSTR4-pFast Bac1 were constructed,and transferred into insect cell SF9;protein subcellular localization vector CsSTR2-pCAMBIA1302,CsSTR4-pBI121-GFP successfully were introduced into Agrobacterium GV1301,transformed into onion epidermal cells;promoter expression analysis vector CsSTR2pro-pCAMBIA1301 were constructed,successfully introduced it into Agrobacterium GV1301 and infected with Arabidopsis plants.3.By comparing the amino acid sequence of the protein,Arabidopsis thaliana was used as control,CsSTR1,CsSTR2,CsSTR3 and CsSTR4 were located on different small branches,CsSTR1 and AT2G41290,CsSTR2 and AT1G08470,AT5G22020,CsSTR3 and AT3G59530,CsSTR4 and AT3G57030 is highly similar.Protein structure prediction showed that CsSTR and RsSTR have low homology.Sequence analysis of the gene family show that STR gene existed as a multigene family in tea trees,and 17 members of the STR gene family were identified by genome-wide analysis.Through the phylogenetic tree analysis of Arabidopsis thaliana and rice STR members,they can be divided into five subfamilies.4.Using the online bioinformatics analysis website,the subcellular localization analysis of CsSTR protein showed that CsSTR2 was mainly located outside the plant tonoplast and CsSTR4 was mainly located in the tonoplast.The subcellular localization vectors CsSTR2-pCAMBIA1302 and CsSTR4-pBI121-GFP were constructed,and the recombinant vector were introduced into the onion epidermal cells by Agrobacterium infection technique.Subcellular localization results indicated that CsSTR2 was mainly localized in plant cell membrane or cytoplasm,and CsSTR4 was mainly located in cytoplasm.The result is in line with the biological information prediction.5.Heterologous expression and enzyme activity detection of CsSTR:The codon preference of CsSTR2 and CsSTR4 was analyzed and inserting the two gene coding sequences into the expression vector pFast Bac1,and transferring them into insect cells for induction,CsSTR2 and CsSTR4 mostly were present in the form of inclusion bodies.A small amount of soluble protein was present in the supernatant of CsSTR4.CDS of CsSTR3was inserted into the expression vector pET-30a（+）and transferred into the BL21（DE3）prokaryotic expression strain,and a soluble fusion protein was obtained in the supernatant.The catalytic products of CsSTR3 and CsSTR4 soluble fusion proteins were detected by HPLC,the two fusion proteins have the catalytic activity of STR enzyme.6.The expression pattern of CsSTR gene in tea tree showed that the CsSTR1 gene was up-regulated and down-regulated under low temperature induction,and the amount of change was obvious.Under drought treatment,it was up-regulated under drought treatment and reached the maximum at 6 h and down-regulated to the pre-control level;in the case of high salt,the expression rapidly reached the maximum value,and then decreased back to the pre-treatment level;under hormone treatment,MeJA caused the expression level of CsSTR1 to be up-regulated rapidly,but the change was not obvious;under ABA treatment,it was firstly adjusted and then rapidly up-regulation;after 6 h of GA₃ treatment,the expression level was suddenly increased to the maximum value of 4 times of the control,and then decreased to the pre-treatment level;under the wound treatment,the expression level was rapidly increased by 12 h to reach the maximum value of 8 times of the control group and then reduced to the pre-treatment level.For CsSTR2 drought,high salt and hormone ABA treatment,there was no significant change in gene expression;under MeJA and SA treatment,the expression level was continuously up-regulated;GA₃ treatment reduced the expression level;under pest feeding and trauma,the expression level was down-regulated first and up-regulated later.The effect of different factors on the expression of CsSTR3 gene was down-regulated at 0-24 h at low temperature and 3.8-fold at 48 h,and was up-regulated to 3.6-fold in the control group and then down-regulated to the pre-treatment level.SA treatment kept the expression level up-regulated;pests fed up to increase the expression level,but the time-varying expression level was up-regulated;the initial expression level of CsSTR4 in tea trees was low,and the expression level was rapidly up-regulated at low temperature and then down-regulated to 12 h.After falling to the lowest value and then returning to the pre-treatment level,the expression level suddenly increased under drought and pest feeding treatment,and there was no significant change in gene expression under hormone treatment.The wound treatment caused the expression level to decrease at 6-24 h.