Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum
<p>Multigene reconstruction to enhance dendrobine production. (<b>A</b>) Physical map of the multigene constructs used for integration and expression of the synthetic operons. The selected target genes are depicted as light blue boxes. Promotor <span class="html-italic">Prrn</span> is shown in dark yellow and terminator <span class="html-italic">TrbcL</span> in orange. The <span class="html-italic">HPTII</span> selectable marker gene for transformation is represented as a light yellow box. An intercistronic expression element (<span class="html-italic">IEE</span>) was put between <span class="html-italic">STR1</span> and <span class="html-italic">CYP94C1</span> operons to ensure the downstream cistron expression under the same promotor. (<b>B</b>) <span class="html-italic">Not</span> I-digestion analysis of pYLTAC380H-multigene (<span class="html-italic">EV</span>, <span class="html-italic">TG</span>, <span class="html-italic">FG</span>, and <span class="html-italic">SG</span>) constructs. M: DNA ladder marker. (<b>C</b>) Dendrobine content in <span class="html-italic">D. catenatum</span> leaves infiltrated with <span class="html-italic">Agrobacterium tumefaciens</span> carrying multigene constructs. There are three replicates for each sample. (<b>D</b>) The relative expression of each gene in a specific multigene construct. <span class="html-italic">EV</span>: empty vector; <span class="html-italic">TG</span>: two genes; <span class="html-italic">FG</span>: five genes; <span class="html-italic">SG</span>: seven genes. Asterisks indicate significance based on the Student’s <span class="html-italic">t</span>-test. * <span class="html-italic">p</span> ≤ 0.05; ** <span class="html-italic">p</span> ≤ 0.01; *** <span class="html-italic">p</span> ≤ 0.001; **** <span class="html-italic">p</span> ≤ 0.0001.</p> "> Figure 2
<p>Functional verification of <span class="html-italic">MCT</span> in dendrobine synthesis. (<b>A</b>) <span class="html-italic">MCT</span> expression was checked by qRT-PCR with samples being collected 6 h after infiltration (n = 3). (<b>B</b>) Transiently infiltrated <span class="html-italic">D. catenatum</span> leaves were harvested (5 dpi) for dendrobine measurement (n = 3). (<b>C</b>) CRISPRi was performed to knock down <span class="html-italic">MCT</span> expression. The kinase-dead version of Cas9 (dCas9) was used to block transcription in the promotor region of <span class="html-italic">MCT</span>. Empty vector without dCas9 served as the control. <span class="html-italic">MCT</span> knock-down was verified by qRT-PCR, with samples being collected 6 h after infiltration (n = 3). (<b>D</b>) Leave samples were collected at 5 dpi and subjected to dendrobine measurement (n = 3). ** <span class="html-italic">p</span> ≤ 0.01; *** <span class="html-italic">p</span> ≤ 0.001; **** <span class="html-italic">p</span> ≤ 0.0001.</p> "> Figure 3
<p>Generation and identification of multigene-transgenic plants. (<b>A</b>) Transgenic <span class="html-italic">D. catenatum</span> plantlets of <span class="html-italic">SG</span>-multigene (11-month-old) and <span class="html-italic">EV</span> control (13-month-old). (<b>B</b>) Analysis of the <span class="html-italic">hptII</span> transgene in <span class="html-italic">SG</span>-multigene and <span class="html-italic">EV</span> control transgenic <span class="html-italic">D. catematum</span> plantlets. DNA isolated from wild-type plants was used as the negative control “−”, while the <span class="html-italic">hptII</span> gene in the <span class="html-italic">EV</span> plasmid was used as the positive control “+”. <span class="html-italic">DcActin</span> was PCR amplified to demonstrate an equal amount of loading. <span class="html-italic">nptII</span> was amplified to avoid bacterial contamination. (<b>C</b>) <span class="html-italic">FPPS</span> expression was checked to demonstrate activation of the dendrobine synthesis pathway. <span class="html-italic">EV</span> transgenic <span class="html-italic">D. catenatum</span> served as the control (Ctrl). (<b>D</b>) <span class="html-italic">SG</span>-transgenic <span class="html-italic">D. catenatum</span> grown in pine-bark pots for 10 months. (<b>E</b>) Molecular characterization of the <span class="html-italic">hptII</span> transgene in <span class="html-italic">SG</span>-transgenic <span class="html-italic">D. catenatum</span> grown in pine-bark pots. (<b>F</b>) Molecular characterization of the <span class="html-italic">hptII</span> transgene in <span class="html-italic">SG</span>-transgenic <span class="html-italic">Arabidopsis</span>. (<b>G</b>) Expression analysis of individual genes in <span class="html-italic">SG</span>-transgenic <span class="html-italic">Arabidopsis</span> by qRT-PCR. Scale bar in (<b>A</b>) represents 1 cm. **** <span class="html-italic">p</span> ≤ 0.0001 represents significance.</p> "> Figure 4
<p><span class="html-italic">SG</span>-transgenic <span class="html-italic">Arabidopsis</span> tolerant to salinity stress. (<b>A</b>) Plant growth in response to various concentrations of NaCl (0, 100, 120, and 200 mM). (<b>B</b>) Plant growth in terms of fresh weight under 100 mM NaCl for four weeks. (<b>C</b>) Cell damage in terms of MDA release under 100 mM NaCl for four weeks. (<b>D</b>) Representative image showing the transgenic plants growing in earth-pot for one month. (<b>E</b>) Comparison of plant height for the transgenic plants growing in earth-pot for one month. Data are represented as means ± SE from three replicates. *** <span class="html-italic">p</span> ≤ 0.001 and **** <span class="html-italic">p</span> ≤ 0.0001 are of significance compared to <span class="html-italic">EV</span> controls. Scale bar represents 1 cm. The center line in (<b>E</b>) represents median.</p> "> Figure 5
<p><span class="html-italic">SG</span>-transgenic <span class="html-italic">Arabidopsis</span> was tolerant to drought stress. (<b>A</b>) Plant growth in response to different concentrations of PEG6000 (0, 250, 400, and 550 g/L). (<b>B</b>) Plant growth in terms of fresh weight under varied concentrations of PEG6000 for four weeks. (<b>C</b>) Cell damage in terms of MDA release under varied concentrations of PEG6000 for four weeks. * <span class="html-italic">p</span> ≤ 0.05; ** <span class="html-italic">p</span> ≤ 0.01; **** <span class="html-italic">p</span> ≤ 0.0001 represent significance. Scale bar represents 1 cm.</p> "> Figure 6
<p>Dendrobine synthesis-related gene screening by Venn diagram and KEGG analysis. (<b>A</b>) Venn distribution of DEGs for transcriptomes of different tissues (stem vs. root; leaf vs. root) from three <span class="html-italic">Dendrobium</span> species (<span class="html-italic">D. houshanense</span>; <span class="html-italic">D. catenatum</span>, and <span class="html-italic">D. moniliforme</span>). (<b>B</b>) KEGG pathway enrichment of 648 DEGs from (<b>A</b>). The x-axis represents the enrichment ratio and the y-axis represents the pathway name. (<b>C</b>) Venn diagram representation of the number of DEGs in samples from protocorm-like bodies (PLBs), samples under <span class="html-italic">MF23</span> treatment, and the 648 DEGs in (<b>A</b>). (<b>D</b>) KEGG pathway enrichment of DEGs from (<b>C</b>).</p> "> Figure 7
<p>Dendrobine synthesis-related hub gene screening by WGCNA. (<b>A</b>) Hierarchical cluster dendrogram showing six expression modules of co-expressed genes. Each leaf in the tree represents an individual gene, with the branch representing a module of highly connected genes. The designated color rows below correspond to module membership. (<b>B</b>) Scale-free fit index at different threshold values (<span class="html-italic">β</span>). Asterisk indicates the selected soft-thresholding power. (<b>C</b>) Heatmap of connectivity of eigengenes. (<b>D</b>) Module-trait correlations and corresponding <span class="html-italic">p</span>-values (in parenthesis). The color in the box indicates −log(<span class="html-italic">P</span>) and the color scale indicates the <span class="html-italic">p</span>-value from the Fisher exact test. Treatment means <span class="html-italic">MF23</span> infection.</p> "> Figure 8
<p>Functional verification of downstream genes in dendrobine synthesis. (<b>A</b>) <span class="html-italic">CMEAO</span> overexpression was verified by qRT-PCR. Samples were collected at 24 h post-infiltration. (<b>B</b>) Transiently infiltrated <span class="html-italic">D. catenatum</span> leaves (5 dpi) were harvested and subjected to dendrobine measurement. Empty vectors are used as controls. (<b>C</b>) qRT-PCR verification of <span class="html-italic">MYB61</span> overexpression in transiently infiltrated one-year-old <span class="html-italic">D. catenatum</span> leaves. (<b>D</b>) Dendrobine content in <span class="html-italic">D. catenatum</span> leaves transiently overexpressing <span class="html-italic">MYB61</span>. Statistical significance was demonstrated as following ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Reconstitution of Multigene for Improved Dendrobine Synthesis
2.2. Overexpression of SG-Multigene Confers Salt and Drought Tolerance to Transgenic Arabidopsis
2.3. Identification of Downstream and Regulatory Genes Related to Dendrobine Synthesis
2.4. Characterization of Novel Genes Associated with Dendrobine Accumulation
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Reagents
4.2. WGCNA and Modular Characterization
4.3. Vector Construction
4.4. Genetic Transformation
4.5. Metabolic Profiling
4.6. qRT-PCR Validation of Gene Expression
4.7. Data Analysis
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Zhao, M.; Zhao, Y.; Yang, Z.; Ming, F.; Li, J.; Kong, D.; Wang, Y.; Chen, P.; Wang, M.; Wang, Z. Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum. Int. J. Mol. Sci. 2024, 25, 397. https://doi.org/10.3390/ijms25010397
Zhao M, Zhao Y, Yang Z, Ming F, Li J, Kong D, Wang Y, Chen P, Wang M, Wang Z. Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum. International Journal of Molecular Sciences. 2024; 25(1):397. https://doi.org/10.3390/ijms25010397
Chicago/Turabian StyleZhao, Meili, Yanchang Zhao, Zhenyu Yang, Feng Ming, Jian Li, Demin Kong, Yu Wang, Peng Chen, Meina Wang, and Zhicai Wang. 2024. "Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum" International Journal of Molecular Sciences 25, no. 1: 397. https://doi.org/10.3390/ijms25010397