| Objective:Codonopsis pilosula polysaccharide is an important active ingredient and biomarker of Codonopsis pilosula,and is also one of the authentic characteristics of Codonopsis pilosula.Codonopsis pilosula polysaccharide has the effects of lowering blood sugar,antioxidation,andanti-tumor.The polysaccharide content of Codonopsis pilosula in Shanxi genuine medicinal material Ludangshen is significantly higher than that in other producing areas.The polysaccharide of Codonopsis pilosula is mostly fructan,accounting for about 50% to 60%.Fructans with different molecular weights and sizes have different effects.The molecular weight of fructan is determined by its synthesis and degradation pathways.Currently,the key enzyme gene for codonopsis pilosula fructosyltransferase,Cp1-SST,has been cloned.However,the key enzyme genes involved in the degradation of codonopsis pilosula fructan have not yet been found.Therefore,searching for the key enzyme genes of codonopsis pilosula fructan degradation will lay the foundation for clarifying the mechanism of fructan synthesis,and does not provide a theoretical basis for gene targeted breeding based on fructan.In this paper,the molecular mechanism of the regulation of codonopsis pilosula polysaccharide synthesis by fructan exohydrolase gene was elucidated through RACE technology,analysis of gene expression patterns under different stress conditions,and analysis of fructan content and gene overexpression based on callus genetic transformation system.Methods:1.The full length of Cp1-FEH and Cp6-FEH sequences of Codonopsis pilosula fructan exohydrolase genes were cloned using RACE technology.Bioinformatics analysis was performed on Cp1-FEH and Cp6-FEH genes.2.The seedlings of Codonopsis lanceolata were treated with low temperature,drought,temperature difference,and iron stress.Combining q RT-PCR and HPGPC techniques,the correlation between the expression of Codonopsis lanceolata fructan exohydrolase genes Cp1-FEH,Cp6-FEH,and the content of Codonopsis lanceolata polysaccharide under stress conditions was analyzed.3.Using the genetic transformation system of Codonopsis pilosula callus,overexpression of Codonopsis pilosula fructan exohydrolase genes Cp1-FEH and Cp6-FEH was performed.Combined with q RT-PCR technology,the effect of overexpression of Codonopsis pilosula fructan exohydrolase genes Cp1-FEH and Cp6-FEH on the expression of genes related to Codonopsis pilosula fructan synthesis and metabolism was analyzed.Results:1.The results of the RACE experiment showed that the full length of Cp1-FEH and Cp6-FEH sequences of Codonopsis pilosula fructan exohydrolase genes were successfully cloned,with an ORF of 1731 bp and encoding 576 amino acids.The ORF of the Cp6-FEH gene is 1725 bp,encoding 574 amino acids.Both belong to the glycoside hydrolase 32 family.2.Using different tissues of Codonopsis pilosula as materials for q RT-PCR analysis,it was found that Cp1-FEH and Cp6-FEH had the highest relative gene expression in the root.The results showed that Cp1-FEH gene can respond to drought stress,low temperature stress,temperature difference stress,iron deficiency stress,and iron deficiency stress in Codonopsis pilosula seedlings under different environmental stresses.The Cp6-FEH gene responds to drought stress,low temperature stress,temperature difference stress,and iron deficiency stress,but does not respond to iron deficiency stress.At the same time,it was found that there was a linear and negative correlation between Cp1-FEH gene expression and fructose content under drought and temperature difference stresses.The gene expression of Cp1-FEH under 4 ℃ cold stress showed a linear and positive correlation with fructan content.There was no correlation between the expression of Cp1-FEH gene and the content of fructan under iron stress.The gene expression of Cp6-FEH under drought treatment was linearly and negatively correlated with fructan content.There was no correlation between the expression of Cp6-FEH gene and the content of fructan under 4℃cold stress and temperature difference stress.The gene expression of Cp6-FEH was linearly and positively correlated with the content of fructan under iron difference stress.3.Plant fusion expression vectors p CAMBIA3301-Cp1-FEH-e GFP and p CAMBIA3301-Cp6-FEH-e GFP were successfully constructed.Positive overexpression calli were obtained through PCR identification.After overexpression of the codonopsis pilosula fructan exohydrolase gene Cp1-FEH,the expression of Cp6-FEH and Cp1-SST genes significantly decreased.The expression levels of Cp NI and Cp SS genes remained basically unchanged.After overexpression of Cp6-FEH gene,the expression levels of Cp1-FEH and Cp NI genes remained basically unchanged,while the expression levels of Cp SS and Cp1-SST genes were higher than those of the control group.Conclusion:1.The full-length sequences of the fructose exohydrolase genes Cp1-FEH and Cp6-FEH were cloned,and the ORF of Cp1-FEH was 1731 bp,encoding 576 amino acids.The Cp6-FEH gene ORF has a total length of 1725 bp and encodes 574 amino acids.The Cp1-FEH gene responds to drought stress,low temperature stress,temperature difference stress,iron deficiency stress,and iron deficiency stress.The Cp6-FEH gene responds to drought stress,low temperature stress,temperature difference stress,and iron deficiency stress,but does not respond to iron deficiency stress.2.The Cp1-FEH gene responds to drought,low temperature,temperature difference,iron deficiency,and iron deficiency stresses.The Cp6-FEH gene responds to drought,low temperature,temperature difference,and iron stress,but does not respond to iron deficiency stress.At the same time,the content of Fructan and its correlation with gene expression under drought,low temperature,temperature difference and iron stress were measured.The results showed that Cp1-FEH under drought,temperature difference and low temperature stress might affect the synthesis and metabolism of Fructan in the roots of Codonopsis pilosula seedlings.Cp6-FEH may affect the synthesis and metabolism of Fructan in the roots of Codonopsis pilosula seedlings under drought stress and iron difference stress.3.After overexpression of Cp1-FEH gene,the expression of Cp1-SST gene is significantly reduced,that is,overexpression of Cp1-FEH gene may reduce the synthesis of Fructan.Overexpression of Cp6-FEH gene will promote the expression of Cp1-SST gene,that is,overexpression of Cp6-FEH gene may promote the synthesis of Fructan. |
