Genome-wide identification and characterization of active ingredients related β-Glucosidases in Dendrobium catenatum

Abstract

Background Dendrobium catenatum/D. officinale (here after D. catenatum), a well-known economically important traditional medicinal herb, produces a variety of bioactive metabolites including polysaccharides, alkaloids, and flavonoids with excellent pharmacological and clinical values. Although many genes associated with the biosynthesis of medicinal components have been cloned and characterized, the biosynthetic pathway, especially the downstream and regulatory pathway of major medicinal components in the herb, is far from clear. β-glucosidases (BGLUs) comprise a diverse group of enzymes that widely exist in plants and play essential functions in cell wall modification, defense response, phytohormone signaling, secondary metabolism, herbivore resistance, and scent release by hydrolyzing β-D-glycosidic bond from a carbohydrate moiety. The recent release of the chromosome-level reference genome of D. catenatum enables the characterization of gene families. Although the genome-wide analysis of the BGLU gene family has been successfully conducted in various plants, no systematic analysis is available for the D. catenatum. We previously isolated DcBGLU2 in the BGLU family as a key regulator for polysaccharide biosynthesis in D. catenatum. Yet, the exact number of DcBGLUs in the D. catenatum genome and their possible roles in bioactive compound production deserve more attention. Results To investigate the role of BGLUs in active metabolites production, 22 BGLUs (DcBGLU1-22) of the glycoside hydrolase family 1 (GH1) were identified from D. catenatum genome. Protein prediction showed that most of the DcBGLUs were acidic and phylogenetic analysis classified the family into four distinct clusters. The sequence alignments revealed several conserved motifs among the DcBGLU proteins and analyses of the putative signal peptides and N-glycosylation site revealed that the majority of DcBGLU members dually targeted to the vacuole and/or chloroplast. Organ-specific expression profiles and specific responses to MeJA and MF23 were also determined. Furthermore, four DcBGLUs were selected to test their involvement in metabolism regulation. Overexpression of DcBGLU2, 6, 8, and 13 significantly increased contents of flavonoid, reducing-polysaccharide, alkaloid and soluble-polysaccharide, respectively. Conclusion The genome-wide systematic analysis identified candidate DcBGLU genes with possible roles in medicinal metabolites production and laid a theoretical foundation for further functional characterization and molecular breeding of D. catenatum.

Analysis of the Glycoside Hydrolase Family 1 from Wild Jujube Reveals Genes Involved in the Degradation of Jujuboside A

“…Others are localized in plasma, mitochondria, and vacuolar. The predicated location of these proteins reflects their diversified functions in wild jujube, as has been reported for other species [ 46 , 47 ].…”

Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum

“…Deactivated Streptococcus pyogenes Cas9 (d Sp Cas9) was introduced into the promotor region of MCT by sgRNA (5′-GATGCAAGAAAGAAAACCTA-3′) to knock down gene expression. The transient expression of dCas9 in D. catenatum leaves was performed following our previous protocol [ 21 ]. Agrobacterium EHA105 cells were transformed with plasmids containing the gene of interest via electroporation (2.5 kv, cuvettes with 0.1 cm gap).…”

“…The suspensions were incubated at room temperature for three hours. For transient expression, the suspensions were slowly infiltrated into the abaxial side of the leaves of one-year-old D. catenatum [ 21 ] or one-month-old N. benthamiana using a needleless syringe. Each group consisted of at least ten plants.…”

“…Similarly, while dendrobine content was detected high in D. nobile , it was only detected occasionally in D. catenatum [ 17 ]. In earlier studies, we sequenced the genome of D. catenatum [ 18 ], analyzed the biosynthetic pathways of dendrobine [ 19 ], identified alkaloid biosynthesis associated genes including STR1 and β-Glucosidases ( BGLUs ), and verified BGLU2 , 6 , 8 , 13 as regulators of alkaloid production [ 20 , 21 ]. Here, we pursued a novel synthetic biology approach for the high level production of dendrobine in D. catenatum leaves.…”