Study On The Effects Of The Content Of Polysaccharides And Mechanism Under The Different Temperature Between Day And Night In Dendrobium Officinale PLBS

Dendrobium officinale, a genus of Orchidaceae, has a very unique medicinal value, In this study, the protocorm of Dendrobium officinale was used as material,the best culture condition for PLBS multiplication, conservation and polysaccharides synthesize were investigated, and liquid culture system was established to determine the content of polysaccharides under the different temperature between day and night. on this basis, cloned two key glycosyltransferases genes— DoGAUTl and DoPGSIP6 in the process of polysaccharide biosynthesis. Furthermore, analysis of the quantitative expression of genes DoGAUTl and DoPGSIP6 in the in vitro cultures was conducted under the different temperature between day and night. The main results were showed as follows:1 Optimization of in vitro culture conditions in Dendrobium officinaleThe protocorm of Dendrobium officinale was used as meterial for proliferation and preservation of in vitro culture condition. ①Single factor test was carried out to compare the effects of combination of 6-BA, coconut water and potato on the proliferation of protocorm, and the result indicated the best medium for proliferation of protocorm in Dendrobium officinale was 1/2MS medium supplemented with 50 g/L smashed potato. Additionally,2.0 mg/L 6-BA was then added to the mix, which showed the enhancement proliferation of protocorm but partly differentiation. ②Comparing the effects of proliferation on protocorm by changing the proportion of KNO3 and KH2PO4 in 1/2 MS. Conclusion illustrated the proliferation coefficient was high but slightly etiolation when KNO3 and KH2PO4 were 0.5 times and 1 times as controlled respectively. The best medium was the control group with 1/2 MS. ③Comparison of effects of different ratio of nitrate nitrogen and ammonium nitrogen on the proliferation of protocorm in Dendrobium officinale, we found that both the medium of 50 g/L homogenates of smashed potato and KNO3 and NH4NO3 were 0.5 times as controlled and the medium of the 50 g/L homogenates of smashed potato was good for the growth of the protocorm..④Comparison of the effect of different concentrations of curcumin and betanin in Dendrobium officinale of in vitro conservation, the result indicated that the curcumin of 0.1-1.0 g/L concentration could restrain the growth of protocorm, while the 0.1 g/L concentration of betanin could have the better effect of preservation. ⑤The induced protocorm of Dendrobium officinale was used as meterial inoculated on the liquid medium contains 50 g/L extract of potato, comparing the biomass change of the culture of Dendrobium officinale at different time periods, the result showed that fresh weight of the protocorm tended as "S" curve in the whole growth cycle and with the rapid growth from 30 to 40 days.2 Measuring the polysaccharide content of in vitro culture under the condition of different temperature difference between day and nightThe induced protocorm of Dendrobium officinale was used as meterials to comparie the polysaccharide content change under the different time, the results indicated that the polysaccharide content also changed as "S" cure in the whole growth cycle. Proper selection the protocorm of Dendrobium officinale at the 30th day as main studying materials, comparing the polysaccharide content of the culture of Dendrobium officinale in the different temperature difference between day and night, The result indicated that the content of polysaccharide was the hightest after 2 days at 25 ℃/10 ℃ and the polysaccharide content increased at first and then decreased with the time within a certain time. While the polysaccharide content increased with the increase at 25 ℃/15℃ treatment temperature.Among them, the temperature was the most desirable accumulation of polysaccharide ones. Meanwhile, The result of the temperature at 25 ℃ /20 ℃ in response to the polysaccharide content showed an increasing trend and the temperature of 25 ℃/20℃ treatment condition was less than those at 25 ℃/15℃.3 Cloning and bioinformatical analysis of DoGAUTl and DoPGSIP6 gene from Dendrobium officinaleIn this experiment, the full-length cDNA of the two glycosyltransferase genes DoGAUTl and DoPGSIP6 gene were successfully cloned from the in vitro protocorm in Dendrobium officinale by the technology of homologous cloning combined with RACE. The full-length sequences of cDNAs were 2518 bp and 2010 bp encoding 705 amino acids and 549 amino acids respectively.Their accession number were 1740985 in GenBank. Bioinformatics analysis showed that the protein of DoGAUTl was likely a kind of transmembrane structure, hydrophilic protein without signal peptide sequence, which might be form coil helix structures and located in the golgi. While the protein of DoPGSIP6 was likely a kind of hydrophobicity, transmembrane structure protein and contained a signal peptide, which could not form coil helix structures and located in the plasma membrane.They belonged to glycosyltransferases 8 with the GT-A fold.The position 394-692 amino acid of DoGAUTl had a number of ligand binding sites as well as two metal binding sites,containing a typical GT8_like₁ conserved domain with unknown function. And the position 29-276 amino acid of DoPGSIP6 had a number of multiple substrate binding site, two Mn2+ binding sites and a conserved DXD motif which added a range of different sugars to other sugars, phosphates and proteins, containing a typical GT8_Glycogenin conserved domain. In addition, the DoPGSIP6 in the position of 522-522 contained a CCC1_like conserved domain which had the effect of nitrogen fixation. Amino acid sequence aligenment showed that DoGAUT1 and DoPGSIP6 displayed the closest evolutionary relationships to those of Brachypodium distachyon and Glycine max, as far as Solanum lycopersicum and Citrus sinensis.4 The analysis of the quantitative expression of DoGAUT1 and DoPGSIP6 under day-and-night temperature differences in Dendrobium officinaleUsing the 18S rRNA as the reference gene, the quantitative expression levels of DoGAUT1 and DoPGSIP6 were analysed under day-and-night temperature differences in Dendrobium officinale.The results showed that the expression level of DoGAUT1 and DoPGSIP6 were positively related to the polysaccharide content at 25 ℃/15℃,and DoPGSIP6 gene had the the highest polysaccharide content and relative expression level with the day-and-night temperature difference,of 15℃ for two days.