Establishment Of Suspension Cells In Chinese Jujube (Ziziphus Jujuba Mill.) And Its Application In CAMP Synthesis And Regulation

Suspension cells is a good model system for the study of single cell and intercellular level,and it could be applicated to effectively produce metabolites such as alkaloids,proteins,sugars and spice natural pigments,which has been established in many plant species.However,the establishment of Chinese jujube suspension cells has not been reported.Cyclic adenosine monophosphate(cAMP)plays an extensive role in plant growth and environmental adaptability.The content of cAMP in mature fruit of Chinese jujube is the highest among the reported plant materials,but the mechanism of which factors affected its synthesis is not clear.In the current study,the immature embryos and leaves of Chinese jujube tissue culture were used to study the effects of genotypes,the ratio of exogenous hormones and culture conditions on callus induction.Then,according to these foundations,a stable suspension cell cultivating system of Chinese jujube was successfully established for the first time,which is of great significance for the genetic transformation of Chinese jujube and the development and utilization of secondary metabolites.Furthermore,we took the suspension cells as experimental materials to study the regulating mechanism of cAMP synthesis which could provide the theoretical basis for the regulation mechanism of cAMP metabolism in Chinese jujube.The main results are as follows:1.The types of explants,genotypes and components of culture medium all have great influence on the status of callus formation of Chinese jujube.Taking leaves and immature embryos as materials,we carried out callus induction and proliferation of Chinese jujube.Results showed that most of the leaf callus were light green,the texture was compact and hard,and they were not easy to disperse.However,some of the callus induced by immature embryos were different according to different cultivars,and some of them were browned gradually,and some of them formed soft embryogenic calli after many times of subculture.Through the screening for the ratio of sugars and exogenous hormones,we found the most suitable induction medium of callus was MS+Maltose 20 g/L+Agar 5.5 g/L+NAA 0.05 mg/L+TDZ 0.3 mg/L.The most suitable medium for callus proliferation was MS+Maltose 20 g/L+Agar 5.5 g/L+NAA 0.5 mg/L+TDZ 3 mg/L.2.Effects of genotypes and components of culture medium on Chinese jujube suspension cells.There were great differences among different genotypes of Chinese jujube embryo cells in suspension cultivation.Only ’Wuhezao76’ performed well in suspension cultivation,which can maintained stable and uniform growth in the process of subculture.Most of the tested embryo cells browned after 2-3 days of suspension cultivation,and then died.After screening the ratio of sugars and exogenous hormones,we found the most suitable suspension cell cultivating medium was MS+Maltose 20 g/L+NAA 0.5 mg/L+TDZ 3.0 mg/L.3.Taking the embryogenic callus of ’wuhezao76’ as experimental material,a stable Chinese jujube suspension cells was established.The most suitable medium for initiatial and proliferation cultivation was MS+Maltose 20 g/L+NAA 0.5 mg/L+TDZ 3.0 mg/L.The suspension cells was cultivated at the rotational speed of 150 rpm and subcultivated every 7 days.The large cell mass was removed in each subcultivation,and the stable suspension cell line could be established after 2 months of cultivation.The growth curve of suspension cells showed "S" shape,so the growth state of suspension cells was the best at 2-4 days after subcultivation.4.Taking suspension cell lines as materials,we studied the metabolic regulating mechanism of cAMP in Chinese jujube.It was found that cAMP and microtubule play an important role in the Ca2+permeable channels,such as the expression of ZjCNGC2 could be regualted by cAMP and microtubule.In Chinese jujube cells,cAMP interacted with Ca2+ and activated by each other.cAMP could promote the influx of Ca2+by activating the ZjCNGC2,while Ca2+can promote the synthesis of cAMP by activating the expression of ZjAC2 and ZjAC5.ZjMAPK2 is the downstream gene of cAMP,which is not regulated by Ca2+,but regulated by cAMP.Microtubules also play an important role in the cAMP signal pathway.Anti-microtubule drug treatment has the same regulatory function as cAMP,which could also promote the influx of Ca2+ by activating the ZjCNGC2,and then Ca2+ can promote the synthesis of cAMP by activating the expression of ZjAC2 and ZjAC5.