Effect Of Nitrogen On Gene Expression In Tissue And Cell Of Azadirachta Indica (A. JUSS)

Neem(Azadirachta indica A.Juss) is a tropical plant with abundantly bioactive components, and azadirachtin is the strongest in the bioactive components. Owing to the limitation of its narrow distribution area, long growth cycle and low reproduction rate, low yield of natural neem metabolite from the seed extract restricts its wider application. Biological technology is adopted to improve that problem for the factory production of neem secondary metabolite. MS medium is the most conductive to neem tissue culture in common medium. The molar ratio of nitrate nitrogen to ammonium nitrogen is 2:1 in MS medium. Azadirachtin all accumulated at the intracellar when the molar ratio of nitrate nitrogen to ammonium nitrogen was 3:1, and secreted to the outside of the cell when the molar ratio of nitrate nitrogen to ammonium nitrogen was 4:1.In this paper, we cultured the callus and suspended cells inducted from neem leaves based on the modified MS medium with the molar ratio of 2:1,3:1 and 4:1 of nitrate nitrogen to ammonium nitrogen, respectively,. On 25 days after subculturing above plant tissues, we determined their biomass, soluble protein content, azadirachtin content, limonoids content and chlorophyll content. In addition, their transcriptome profiling was used to reveal gene expression differences among those treatments by high throughput sequencing.When the NO3--N:NH4+-N=4:1, the biomass of callus and suspension cells were maximum,25.85 g/L and 92.13 g/L; the soluble protein, azadirachtin, limonoids and chlorophyll in callus reached also the highest content with 12.67 mg/g,37.55 μg/g,116.10 μg/g and 30.24 mg/kg, respectively; while the NO3--N:NH4+-N=3:1, the contents of soluble protein, azadirachtin, limonoids and chlorophyll in suspension cells played the best performation with 10.18 mg/g,35.61 μg/g,123.20 μg/g and 36.78 mg/kg, respectively.The results also show that the effect of the content and the form of nitrogen source on biosynthesis of terpenoids precursor was small, only reflecting in two key enzymes of HMGR reductase and IPPI isomerase; that effects on the biosynthesis of flavonoids substances were great, mainly reflecting in the activity regulation of some oxidoreductase, transferase and isomerase; and the biosynthesis process of the sterol, cytochrome P450, ribosomal subunits, and so on. Therefore, the change of nitrogen in culture system is an important influence for the biosynthesis metabolism of neem.