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Acta Pharmaceutica Sinica ; (12): 335-344, 2020.
Article in Chinese | WPRIM | ID: wpr-789030


The objective of this research was to clone 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene (MoDXR) and its promoter sequence from Morinda officinalis and carry out bioinformatic analysis, cis-acting elements analysis, and prokaryotic expression. On the basis of the MoDXR gene sequence obtained from the M. officinalis transcriptome and with NCBI-ORFfinder analysis, a pair of specific primers were designed, and used for RT-PCR amplification. The promoter region sequence at the 5′ end of MoDXR gene was isolated by the genome walking technique. Localization of MoDXR was carried out by subcellular analysis. The prokaryotic expression plasmid pET-28a-MoDXR was constructed and transfected into Escherichia coli BL21(DE3) chemically-competent cells; the recombiant plasmid expressed fusion protein after the induction by IPTG. The full-length cDNA of MoDXR was 2 015 bp,and open reading frame (ORF) size was 1 425 bp, and it encoded 474 amino acid residues and had a molecular mass of 51.27 kD. Sequence comparison with BlastP to the NCBI database revealed that MoDXR had high sequence similarity with many other DXRs, such as Coffea arabica DXR (CaDXR) and Rauvolfia verticillata DXR (RvDXR). A phylogenetic tree revealed that MoDXR had its closest relationship with DXR from Coffea arabica and Gardenia jasminoides. The subcellular localization revealed that MoDXR protein was located on the chloroplast. Plantcare analysis indicated that the promoter region sequence of MoDXR was 1 493 bp, covering multiple light, stress, and hormone-responsive cis-regulatory elements; protein electrophoresis showed that the expressed protein was the anticipated size. This research lays the foundation for further purification and structural and functional characterization of the MoDXR protein.

Acta Pharmaceutica Sinica ; (12): 160-167, 2020.
Article in Chinese | WPRIM | ID: wpr-780570


In order to explore MYB transcription factors related to developmental processes and secondary metabolism in Morinda officinalis, we analyzed MoMYB expression based on transcriptome data from three tissues (root, stem and leaf). We used this analysis to provide a theoretical foundation for regulating the metabolism of M. officinalis. RNA-seq data along with the five databases including PFAM and plantTFDB and others were used to screen and classify MoMYB, including GO functional annotation and classification, subcellular localization, signal peptide prediction, conserved motif discovery, and comparative phylogenetic analysis. RT-qPCR was carried out to detect tissue-specific expression differences of MoMYB genes. According to transcriptome data, 109 MoMYB sequences were identified and divided into four classes, containing 51 sequences related to R2R3-MYB. Subcellular localization analysis indicated that a majority of sequences were located in nucleus. Blast2GO analysis showed that 109 MoMYB sequences were classified into three major functional ontologies including molecular function (112), biological processes (76) and cellular components (239). The R2-MYB conserved motif of 51 R2R3-MYB sequences possessed three significantly conserved tryptophan residues, whereas a phenylalanine replaced the first tryptophan in R3-MYB. The results of multiple sequence alignment and phylogenetic analysis revealed that the R2R3-MYB was distributed in all subgroups, apart from the S10, S19 and S21 subgroups. RT-qPCR indicated that several R2R3-MYB genes were differentially expressed among the three tissues, and this finding was consistent with transcriptome data. The 109 MoMYB sequences were annotated and divided into different classes, which lays the foundation for further study on MYB transcriptional factors in M. officinalis.

Article in Chinese | WPRIM | ID: wpr-853133


Objective: Sarcandra glabra was recognized as an important research material attributing to its high medicinal value and economic value. However, little information was known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand the molecular basis and further explore genes which related to metabolism and resistance in S. glabra. Methods: A SMART full-length complementary DNA library from the leaves tissue was constructed and characterized to providing the experimental basis for discovery of functional genes of S. glabra. The assembly expressed sequence tag (EST) data were completed by ABI3730 DNA program. A high quality full-length cDNA library was constructed successfully from S. glabra leaves. Results: The titer of library was 1.14×107 pfu/mL and the average length of inserted fragments was 1 000 bp. A total of 221 clones were sequenced from the cDNA library and obtained 177 EST sequences. The EST sequences were assembled into 151 unigenes including 12 contigs and 119 singletons (79%). EST exhibited significant similarity with known putative functional nucleotide sequences in the GenBank database. These genes were mostly involved in cell development, signal transduction, protein synthesis, transcription, stress tolerance response, energy metabolism based on molecular function of GO annotation. Conclusion: This report constructs a full-length-cDNA library and analyzes the bioinformatics of the related EST sequences, and then offers a reference to genomic research of S. glabra.