Effect Of The Fungal Elicitor On The Alkaloid Contents And Its Mechanism In Dendrobium Officinale PLBs

Dendrobium officinale Kimura et Migo belongs to epiphytic perennial herb,which has great ornamental value and medicinal value. In this article, Protocorm-like bodys(PLBs) from Liancheng Fujian province were used as materials to study the optimizing in vitro culture conditions of the PLBs, and the effects of concentration and treated time of fasurium oxysporum elicitor on the alkaloid contents. In addition, two regulatory genes of alkaloid biosynthesis pathway -- DoHDR and DoIspF were cloned and their relative expression levels were analysed.1 The optimization of in vitro culture conditions of PLBs in Dendrobium officinaleUsing PLBs as meterials to optimize in vitro culture conditions. ①Comparing different concentration of salicylic acid, peptone and tomato juice on PLBs proliferation, the results showed that when the concentration of salicylic acid was 2 mg/L the PLBs proliferation was the best, but PLBs growed poorly. Adding 2 mg/L peptone conducives to PLBs proliferation while adding 100 ml/L tomato juice conducived to PLBs proliferation, and PLBs growed well.②Comparing several groups of different organic additives on the PLBs proliferation, the adopted additives were respectively, bananas and potatoes, apples, and potatoes, bananas and apples, tomatoes and potatoes.The results showed that add 50 g/L potato promotes proliferation best. ③According to the comparation of different containers and culturing modes of PLBs proliferation, the results showed that using liquid-quivering culturing in Erienmeyer flask was best.2 The effects of fungal elicitors on total alkaloid content of PLBsBased on experiments in last chapter we had got large numbers of PLBs, using these as material, comparing the influence of Fusarium oxysporum elicitor on PLBs total alkaloids in different concentrations and different treats time. As a consequence of this, when elicitor concentrations were low (100 mg/L,200 mg/L), total alkaloid content with the increase of treating time was downward trend after rising first, while elicitors concentrations was higher (500 mg/L), total alkaloid was first rise after falling. When concentrations of elicitors were higher (1000 mg/L), total alkaloid synthesis was inhibited, the content was low. The most suitable condition for alkaloid biosynthesis and accumulation was adding 200 mg/L elicitor and treated for three days, the total alkaloid content was the highest.3 Cloning and bioinformatics analysis of HDR and IspF gene from Dendrobium officinaleUsing PLBs as the material, two MEP pathway related genes were got(DoHDR and DoIspF) by combining the technology of homology cloning and RACE. The full length of DoHDR was 1784 bp, containing the complete ORF which length was 1382 bp, encoding 460 amino acids, bioinformatics analysis showed that the protein encoded by this gene was a stable hydrophilic acidic protein, did not have a transmembrane domain, did not contain signal peptide. The secondary structure prediction showed that the protein contained mainly α-helix, β-folding and random coil. The protein contains 20 phosphorylation sites, including seven serine (Ser) phosphorylation site,7 threonine (Thr) sites,6 tyrosine sites (Tyr). This protein was most likely located in endoplasmic reticulum, and belongs to lytB_ispH superfamily, the protein’s affiliation was most close to monocots Phalaenopsis.The full length of DolspF is 891 which contained 785 bp ORF, encoding 260 amino acids, the protein encoded by the gene was an unstable water-soluble protein, containing no transmembrane and signal peptide, the most likely location was chloroplast thylakoid membrane, secondary structure prediction result showed that the protein contains more random coil, and to a lesser a-helix and β-folding. The protein contained 15 protein phosphorylation sites, including 9 serine (Ser) sites,5 threonine (Thr) sites and 1 tyrosine (Tyr) site. Protein functional site prediction results showed that the protein belongs to MECDP synthase superfamily, the protein’s affiliation was most close to Zea mays which belongs to Monocotyledoneae.4. The analysis of relative quantitative expression levels of DoHDR gene and DolspF gene under the treatment of fungal elicitor① Analysis of relative expression of DoHDR under the treatment of Fusarium oxysporum elicitor. When the concentration of elictor was lower (100 mg/L) or higher (500 mg/L), HDR expression level showed the same trend with the group of without elicitor, it increased with treating time which showed downward trend after rising first, the peak appeared after treatment for 5 days, while elicitor concentration was 200 mg/L, HDR gene still showed downward trend after the first rise, but the peak appeared after treatment for 3 days, while elicitor concentration was too high (1000 mg/L), HDR gene expression was inhibited, the expression trend matched to total alkaloid content generally, HDR gene was positively correlated with the alkaloid content. ②Analysis of relative expression of DoIspF under the treatment of Fusarium oxysporum elicitor. DolspF gene expression gradually increased with time without elicitors, when elicitors concentration was 100 mg/L,200 mg/L or 500 mg/L DolspF gene relative expression first increased and then decreased, when elicitor concentration was 1000 mg/L, the gene expression showed a gradual decline substantially.