Studies On Proliferation And Changes Of Catechins Contents Of In Vitro Cultures And Metabolic Mechanism Of Catechins In Camellia Nitidissima

Calli and somatic embryonic cultures were used as materials to do the following studies:optimizing of propagation of calli and somatic embryonic cultures,establishment of suspension cell line and its cell growth curve,the effects of culture condition on the content of catechins from calli and somatic embryonic cultures and expression and correlation analysis of DFR,LAR and PPO in calli under different treatments.The purpose of the above studies was to provide material basis and theoretical guidance for deep development of secondary metabolic products such as catechins.1.Optimizing of propagation of calli and somatic embryonic culturesCalli rich in catechins were used as materials to study the effects of medium volume,weight of calli and culture mode on the propagation of them.The results followed.Suitable condition for propagation of calli was to culture 0.5 g calli in 45 mL MS medium supplented with 4.0 mg/L 6-BA and 0.6 mg/L 2,4-D and to culture them in the order as solid medium-liquid medium-solid medium.Somatic embryonic cultures growing rapidly were used as materials to study the effects of carbon source combination,medium volume,weight of somatic embryonic cultures and culture mode on the propagation of them.The results followed.Suitable condition for propagation of somatic embryonic cultures was to culture 0.5 or 1.0 g somatic embryonic cultures in 45 mL MS medium supplented with 40 g/L saccharose and 20 g/L sorbitol and to culture them in the order as solid medium-solid medium-liquid medium.2.Establishment of suspension cell line and its maintainingStem,leaf,anther and somatic embryo of Camellia nitidissima were used as materials in this study.With the methods of tissue culture and microscopic observation,induction of friable callus and establishment of suspension cell lines were conducted.The study showed the optimal process to obtain friable callus from C.nitidissima chi.According to this work,the slices of somatic embryo were cultured on the MS medium supplemented with 0.5 mg/L KT,8.0 mg/L NAA,5 mg/L AgNO3 and 30 g/L sucrose under dark condition to induce callus.The newly induced calli were separated from somatic embryo and were cultured on callus inducing medium for three months.Finally,the friable calli were subcultured on the media containing inositol or AgNO3 alternatively;According to analysis of variance,the content of sucrose,inositol and AgNO3 significantly affected the cell density in suspension cultured liquid medium of C.nitidissima Chi and the lighting condition has significant interaction with the last two;The optimal process to establish suspension cell line of Camellia nitidissima was as following:Culture the slices of somatic embryo on the MS medium supplemented with 0.5 mg/L KT,8.0 mg/L NAA,5 mg/L AgNO3 and 30 g/L sucrose under dark condition;Separate newly induced calli from somatic embryo and cultured them on callus inducing medium for three months;Culture the friable calli alternatively in liquid proliferation medium containing 100mg/L mositol or 2.5 mg/L AgNO3 under dark condition.Culturing time was studies during the alternative culture mentioned above.According to the result,suitable culture period for primary culture in liquid medium is 20 d and that suitable culture period for subculture in liquid medium is 10 d.3.The effects of culture condition on the content of catechins from calli and somatic embryonic culturesCalli rich in catechins and somatic embryonic cultures growing rapidly were used as materials to study the effects of light,hormones,carbon source and phenylalanine on the propogation and catechins’content of the two.The results were as following.Light,6-BA,2,4-D,6-B A X 2,4-D,type of carbon source、additive amount of carbon,type of carbon source X additive amount of carbon and additive amount of phenylalanine didn’t have any significant effects on the growth of calli but had very significant effects on the content of catechins.Light,6-BA,additive amount of carbon and additive amount of phenylalanine had significant effects on the growth of somatic embryonic cultures;Light,6-BA,NAA,type of carbon source、additive amount of carbon,type of carbon source X additive amount of carbon and additive amount of phenylalanine had very significant effects on the content of catechins in somatic embryonic cultures.Suitable material,medium and light condition for in vitro production of catechins were as following:Culturing calli on MS medium supplented with 4.0 mg/L 6-BA,0.6 mg/L 2,4-D and 0.6608 g/L phenylalanine under fluorescent lamp.4.Cloning and bioinformatics analysis of LAR from calliHomologous cloning and RCAE(rapid amplification of cDNA ends)technology were used to clone LAR gene from calli rich in catechins of Camellia nitidissima.A series of tools were used to do the bioinformatics analysis.The results followed.LAR protein in Camellia nitidissima which might have 4 inside to outside helices and 3 outside to inside helices might be acidity,stable and hydrophobic.Secondary structure of LAR protein in Camellia nitidissima was consisted of a-helix(28.07%),extended strand(27.19%)and random coil(44.74%).Serine phosphorylation might occur on 6,8,114,186,214,231 and 247 these seven protein loci of LAR;Threonine phosphorylation might occur on 107,201 and 245 these three protein loci of LAR;Tyrosine phosphorylation might occur on 163 and these two protein loci of LAR;Among these protein loci,value of 186,107 and 164 is more than 95%.It might be located in cytoplasm;It belongs to NADB Rossmann super family and has NAD(P)binding domain.Sequence of LAR in Camellia nitidissima is very close to that in Camellia sinensis and there is more than one member of LAR in some plants.5.Gene expression and correlation analysis of DFR,LAR and PPO in calliCalli rich in catechins were used as materials to study the variation of DFR gene expression,LAR gene expression,PPO gene expression and content of catechins and the correlation with each other in calli under different light source,hormones,carbon source or phenylalanine treatments for 30 d.The results were as follows.All the above four detecting items reacted to in vitro treatments significantly.Under the above treatments,the expression pattern of DFR gene and LAR gene was very similar.The correlation coefficients between them two under these treatments were between 0.7.10 and 0.889.The correlation of PPO gene expression and content of catechins was significantly negative under different carbon source treatments and their correlation coefficient was-0.696.DFR gene expression was significantly positively related to the content of catechins under different phenylalanine adding quantity treatments and the correlation coefficient was 0.786.LAR gene expression was also significantly positively related to the content of catechins under different phenylalanine adding quantity treatments and the correlation coefficient was 0.564.MS solid medium supplented with 4 mg/L 6-BA,0.6 mg/L 2,4-D,30 g/L sucrose and 0.6608 g/L phenylalanine was suitable for in vitro production of catechins.Content of catechins in calli cultured in this medium for 30 d was about 40.11 mg/g(dry weight).Basing on the above research we concluded that similar to Camellia sinensis there was a close connection between DFR gene and LAR gene during metabolism of catechins in camellia nitidissima and that the increase of expression of PPO gene caused the loss of catechins in camellia nitidissima and that it was an effective way to add suitable amount of phenylalanine for the increase of content of catechins in calli of camellia nitidissima.