Mining And Functional Characterization Of Key Catalase Enzyme Alkaline/Neutral Invertase DcNI4 Involved In Polysaccharide Biosynthesis Pathway In Dendrobium Catenatum

Dendrobium catenatum Wall ex Lindl is a perennial epiphytic herb belonging to the genus Dendrobium of the Orchidaceae family.It has the functions of nourishing the stomach,nourishing fluid,nourishing yin and clearing away heat,lowering blood sugar and blood lipids,anti-tumor,and anti-cancer.Polysaccharides are important active components of D.catenatum.Alkaline/neutral invertase(NI)can irreversibly convert sucrose into fructose and glucose and provide precursors for the synthesis of polysaccharides.The research on the functions of the NI family exploring the synthesis of polysaccharides is of great significance and provides evidence for revealing the pathways of polysaccharide synthesis of D.catenatum.The genome file of D.catenatum was downloaded from the database on the NCBI website.The Dc NI gene family was identified by bioinformatics methods,phylogenetic tree analysis,gene structure and motif analysis,and other bioinformatics analysis,systematically and completely evaluated the officinale the Dc NI gene family of D.catenatum.Through transcriptome data,it was found that Dc NI4 gene is highly expressed in stems,which is consistent with the traits of high polysaccharide content in D.catenatum stems.Therefore,Dc NI4 gene was used as the research object,Dc NI4 gene was cloned and plant overexpression vector and prokaryotic expression vector were constructed.Carried out genetic transformation test and in vitro protein function verification.The following results were obtained:1.Successfully identified and comprehensively analyzed the Dc NI gene family of D.catenatum.Nine Dc NI genes were identified from the D.catenatum genome.Through phylogenetic analysis,Dc NI was divided into two groups,α and β and then into 5 subgroups,which are similar to the NI genes of Phalaenopsis equestris,which belong to the orchid family with a high degree of homology.Dc NI gene family members have different expression levels in different organs(roots,stems,leaves)of D.catenatum,and Dc NI4 gene is highly expressed in stems.Most of their cis-acting elements are related to hormones and adversity stress.They show changes in expression under drought,cold,and jasmonic acid stress,and participate in biotic and abiotic stresses.2.Clone the Dc NI4 gene,with a full length of 1668 bp,encoding 555 amino acids.It was verified by q RT-PCR that the expression of Dc NI4 gene in 6-month-old stems of D.catenatum was significantly higher than that in other parts.Then the plant overexpression vector p CAMBIA1300-Dc NI4-GFP was constructed and then the Agrobacterium-mediated method was used to confirm the expression of Dc NI4 gene in the cytoplasm through the subcellular localization of tobacco transient transformation.Using the same method,Col wild-type Arabidopsis thaliana was successfully infected to obtain Dc NI4 transgenic plants.The results showed that the transgenic plants were not significantly different from the wild-type in morphology,and the polysaccharide content of the transgenic plants was significantly higher than that of the wild-type,which proved that Dc NI4 is in D.catenatum played a role in the synthesis of polysaccharides.3.The p ET32a-Dc NI4 prokaryotic expression vector was successfully constructed.The cells were disrupted and SDS-PAGE gel electrophoresis was performed.The results showed that the proteins were all precipitated.It is speculated that the proteins formed inclusion bodies during the translation process.This study successfully cloned the full-length sequence of the neutral/alkaline invertase 4(Dc NI4)gene from D.catenatum,and successfully verified in vivo that Dc NI4 can irreversibly decompose sucrose to produce fructose and glucose.The research on the function of alkaline/neutral invertase 4 laid the foundation for the analysis of the synthetic pathway of D.catenatum polysaccharide.