The Cloning And Function Analysis Of KGM Synthesis Key Enzymes Gene CDNA In Amorphophallus Konjac

Konjac Glucomannan(KGM)is a unique polysaccharide component in A.konjac and is regarded as the superior quality of human seventh nutrient cellulose.This study was based on konjac transcriptome analysis that two cDNA sequences of KGM synthesis key enzyme genes were identified by BLAST comparison and homology analysis with the transcriptome date.The two cDNAs encoding for GDP-Mannosyl transferase(GMT)and GDP-Mannose Pyrophosphorylase(GMP)were cloned and sequenced.They were named as AkGMT and AkGMP respectively.We constructed the plant expression vectors regulated by 35 S promoter with the cDNA of these two enzyme genes.Then they were transformed into tobacco WS38,BY-2 cells and Arabidopsis thaliana separately by agrobacterium-mediated transformation.The transgenic plants and cells were identified by antibiotic screening and molecular verification.The expression activities of AkGMT and AkGMP genes in heterologous plants and their effects on saccharides metabolism in transgenic plants were explored by studying the changes of their saccharides components.The colorimetric determination was applied to detect the content of water-soluble sugar and reducing sugar in the transgenics and high performance liquid chromatography was applied to extract and analyze the glucose and mannose sugar content in the tissue fluid.The results showed that all the AkGMP transformed tobacco,Arabidopsis thaliana and BY-2 cells have significantly increased in water-soluble sugar and mannose compared with the wild-type control.But they are not in significantly different of reducing sugar and glucose.The AkGMT transformed tobacco,Arabidopsis thaliana and BY-2 cells showed no significant in water-soluble sugar and mannose content,and no significant difference in reducing sugar and glucose content either.It is concluded that the expression of the two genes in the three transformed materials has no significant difference in glucose metabolism but with minor changed mannose increase.The polysaccharides extracts of the three transgenic were hydrolyzed and there was no significant difference compared with the control.These results indicate that AkGMT and AkGMP of A.konjac KGM key enzyme genes in tobacco,Arabidopsis thaliana and BY-2 can affect the anabolism of saccharides in the plant to a certain extent that the synthesis of mannose can be strengthened but there is no KGM synthesized.We concluded that the single function gene expression cannot lead to KGM synthesize in heterogeneous transgenics.In this paper,the changes of sugar components in tobacco plants,tobacco BY-2cells and Arabidopsis thaliana transgenic with AkGMT and AkGMP genes were analyzed,and its effect on saccharides metabolism when they are heterogeneous expression were discussed.It provides a view for further study on the genetic regulation mechanism of saccharides metabolism in plant cells.