| Lutein is ubiquitous in nature,and its antioxidant properties give it a variety of physiological functions.Marigold petals are the main commercial source of extraction of lutein,but marigold is expensive to grow,requires a lot of human and land resources,has low production efficiency,and is restricted by season,climate and region.Plant cell suspension culture has the advantages of short growth cycle,stable genetic characteristics,controllable conditions,no pollution,and can be expanded.However,there are few studies on the molecular mechanism of lutein production and lutein metabolic pathway of marigold plant cells based on plant suspension cell culture technology.In this study,Marigold plants in laboratory planting base were used as materials to study the optimal hormone concentration of callus induced by stem,leaf and flower of Marigold,optimize the initial pH value and initial inoculation amount of suspension cell medium,and the effects of tomato juice and gamma-aminobutyric acid(GABA)alone and combined treatment on lutein in suspension cells.Furthermore,the changes of gene expression of key enzymes in lutein synthesis were further analyzed from the gene level,and the regulation effect of tomato juice and GABA on lutein accumulation in marigold suspension cells was preliminarily clarified.This study can provide theoretical basis for establishing marigold cell suspension lines,gene knockout of marigold cell suspension cells,increasing the content of lutein in marigold cell suspension cells and culture of marigold cell suspension bioreactor.The main research contents are as follows:1.Callus could be induced from stems,leaves and petals of Marigold.The optimal medium concentration of hormone for callus induction from stems was 1.5 mg/L 6-BA+0.1mg/L NAA,and the healing time was 11 d.The optimal medium for callus induction was3.0 mg/L 6-BA+1.0 mg/L NAA,and the healing time was 11 d.The optimal hormone concentration of petal induced callus was 3.0 mg/L 6-BA+0.5 mg/L NAA,and the healing time was 28 d.The growth of petal callus was the best.The optimal dosage of PVP was 0.25mg/L,and the growth of callus was better and the content of lutein was the highest.2.The pH value and initial inoculation amount of marigold suspension cell culture medium were designed by single factor experiment,and the culture conditions of marigold petal cell suspension system were optimized.By measuring the cell biomass,antioxidant enzyme activity and lutein content of marigold petal cell suspension system,the optimum growth conditions of the cells were determined.The experimental results showed that when the initial inoculation amount was 3 g/ bottle,the antioxidant enzyme activity of suspended cells increased,and the lutein content and lutein biomass were higher.This inoculation amount not only had enough growth space for cells,but also did not waste culture medium.When the initial pH value of the medium is 5.8,the cell biomass of Tagetes erecta petal cell suspension culture is the highest,the pH value changes the least,the antioxidant enzyme activity increases,and the lutein content and lutein biomass are the highest.The growth curves of petal cell suspension system of Tagetes erecta were measured under dark and light conditions,respectively.The comparative results showed that the proliferation ability of suspension cells was stronger under dark conditions,but the lutein content and lutein biomass of suspension cells under light conditions were significantly higher than those under dark culture,and the lutein content was 2.39 times that under dark culture conditions.3.Marigold suspension cells were treated with tomato juice with final concentrations of 1 mL/L,2 mL/L,3 mL/L and 4 mL/L and GABA with final concentrations of 0.2 mg/L,0.5 mg/L,0.8 mg/L and 1.1 mg/L,respectively.The results of single factor experiment showed that the antioxidant enzyme activity was higher and the cell biomass,lutein content and lutein biomass were the highest when the concentration of tomato juice was 2 mL/L and GABA was 0.5 g/L.Combined with the results of single factor experiment,a two-factor and three-level response surface experiment was designed for the combined treatment of the two substances.The results showed that the lutein content of 1 mL/L tomato juice +0.8 mL/L GABA concentration combination reached the maximum 8.44 mg/L,SOD activity decreased,POD activity increased and CAT activity increased in suspension cells.SOD activity was inversely proportional to lutein content,POD activity was proportional to tomato juice and GABA concentration,and CAT activity was proportional to lutein content.Multifactor variance analysis of lutein content as response value showed that tomato juice and GABA had interaction on suspension cells.4.Non-reference transcriptome sequencing was performed on CK,2 mL/L tomato juice,0.5 mg/L GABA,1 mL/L tomato juice +0.8 mg/L GABA treatment groups based on Illumina sequencing platform.There were 93381 NR and 84086 NT in the database.Notes to a large number of Unigene in KOG(84141),Swiss-Prot(74241),Uniprot(74168),KEGG(44406),GO(45378).The metabolic pathway enrichment analysis of the statistical differential genes showed that the metabolic pathways regulated by tomato juice and GABA treatment were mainly carotenoid biosynthesis,photosynthesis,biosynthesis of stilbene,diarylheptane and gingerol,biosynthesis of secondary metabolites,DNA replication,etc.In the above metabolic pathways,GABA can promote the up-regulation of lycopene β-cyclase,lycopene epi-cyclase,β-carotene hydroxylase,violet xanthine decycloxygenase genes in the metabolism of carotenoid biosynthesis.Tomato juice can promote the up-regulation of octa lycopene synthetase and octa lycopene dehydrogenase genes in the process of carotenoid biosynthesis.These genes are considered to be the key genes for the increase of lutein content.In summary,both tomato juice and GABA could promote the biosynthesis of lutein.The experimental results of tomato juice combined with GABA showed that the combination of tomato juice and GABA had a promoting effect on the expression of upstream genes,but had a small promoting effect on downstream genes,while GABA had a significant promoting effect on downstream genes.It was concluded that there was a certain synergistic effect between them in the lutein metabolic pathway of Marigold petal cell suspension system. |
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