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Objective:To identify WRKY genes from the transcriptome dataset of Prunella vulgaris by bioinformatics method, and analyze the protein characteristics and expression level of these genes. Method:WRKY transcription factor were identified from the P. vulgaris transcriptome database，their motif，physical and chemical properties，functional annotations，family evolution and expression patterns were analyzed, and their functions were predicted. Result:A total of 23 WRKY transcription factors were identified from P. vulgaris in this study by computational prediction method.Structural analysis found that WRKY proteins contained a highly conserved motif WRKYGQK. Phylogenetic analysis of WRKYs together with the homologous genes from Arabidopsis thaliana could be divided into two groups（group Ⅰ-Ⅱ）. There were 7 members in group Ⅰ，and 16 members in group Ⅱ, group Ⅱ was subdivided into five subgroups，namely group Ⅱb （3 members），Ⅱc（5 members），Ⅱd（3 members），Ⅱe（5 members）. The physical and chemical properties of WRKY protein showed that the amino acid number was between 85 and 599，the molecular weight was between 9 527.5-66 438.45 Da，the theoretical isoelectric point was between 5.01-9.83.Among them, c13719.graph_c0，c32199.graph_c0，c24547.graph_c0，c37881.graph_c0 may play a role in the regulation of secondary metabolitessynthesis of P. vulgaris.And c32199.graph_c0，c26537.graph_c0，c23728.graph_c0 may has an effect in identifying and defensing pathogens in P. vulgaris.The transcriptional profiles of these 23 WRKY genes in various tissues were investigated using transcriptome dataset.The results showed that the expression level of WRKY genes varied significantly in different tissues. Conclusion:This study identifies the organization and transcriptional profiles of PmWRKY genes for the first time, so as to provide the helpful information for further studies of functions of WRKYs.
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Objective: To compare and analyze the transcriptome of ears, leaves and stems of Prunella vulgaris, and excavate the key enzyme genes related to the secondary metabolism biosynthesis of P. vulgaris. Methods: The transcriptome of ears, leaves and stems of P. vulgaris were sequenced by Illumina high-throughput sequencing technology. Additionally biosynthesis related enzyme gene of secondary metabolism were identified from differentially expressed genes. Results: In the transcripts of three different tissues of P. vulgaris, a total of 8 270 Unigenes differed significantly between at least two tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of genes differentially expressed in different tissues showed that the expression of phenylpropanoid biosynthesis genes varied greatly. A total of 31 triterpenoid biosynthesis-related Unigenes, 16 phenolic acid biosynthesis-related Unigenes, and 113 P450s-related Unigenes were identified in the differentially expressed genes. Conclusion: This study provides a basis for the subsequent discovery of functional genes related to the secondary metabolism synthesis pathway of P. vulgaris, and plays a foundation for the regulation of secondary metabolism biosynthesis of P. vulgaris.
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Objective To obtain the transcriptome sequence database and to explore the molecule basis of secondary metabolism biosynthesis in Prunella vulgaris. Methods The high-throughput RNA-seq technology (Illumina HiSeq 2000) was used to conduct a transcriptomic analysis of P. vulgaris ears, stems, and leaves. Unigenes were obtained after assembled by Trinity, and the sequencing results were analyzed with the bioinformatics analysis. Results A total of 77 863 Unigenes were obtained and the average length was 716.72 nt. A total of 41 367 (53.13%) unigenes were annotated by a BLAST similarity search against Nr, Swiss-Prot, COG and other four public sequence databases, and 1 406 Unigenes were assigned to secondary metabolism biosynthesis pathways by the KEGG. Based on the bioinformatic analysis, we found that 60 Unigenes were involved in the triterpenes backbone biosynthesis, 24 Unigenes catalyze synthesis of phenolic acids, and 259 Unigenes might participate in secondary metabolism post-modification. Additionally, 118 unigenes might involve in other secondary metabolism biosynthesis of P. vulgaris. Conclusion The transcriptome data of P. vulgaris from this study provides an important resource for understanding the biosynthesis mechanism of its secondary metabolites, and provides basic information that may aid in metabolic engineering to increase yields secondary metabolites of P. vulgaris.
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Objective To identify the MYB transcription factors that may regulate the biosynthesis of triterpenoids and phenolic acids in Prunella vulgaris. Methods MYB transcription factor were identified from the P. vulgaris transcriptome database, their motif, physical and chemical properties, functional annotation, family evolution and expression patterns were examined. Results A total of 27 MYB transcription factors were identified. c32045.graph_c0 might inhibit the biosynthesis of rosmarinic acid by inhibiting the expression of cinnamic acid 4-hydroxylase gene, thus acting as a transcription suppressor to negatively regulate the biosynthesis of phenolic acids. c26895.graph_c0 might inhibit the biosynthesis of triterpenoids and phenolic acids by regulating the flow of metabolic intermediates in the biosynthesis of triterpenoids and rosmarinic acids. Conclusion The candidate MYB transcription factors related to the biosynthesis of triterpenoids and phenolic acids were obtained. It also laid a foundation for the further study of MYB in regulating of secondary metabolites in P. vulgaris.
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Objective: To clone the key enzyme gene involved in the biosynthesis of esculentoside A(EsA),acetoacetyl-CoA transferase(AACT) gene was cloned from Phytolacca americana for bioinformatics analysis and prokaryotic expression. Method: Total RNA was extracted from the root of P. americana, and then cDNA was synthesized through the reverse transcription. Based on analysis of the transcriptome data of P. americana, the specific primers of PaAACT gene were designed,and the cDNA sequence of PaAACT gene was amplified by polymerase chain reaction(PCR) method. Prokaryotic induction,expression and purification of the target protein were induced through the construction of the prokaryotic expression vector pET-32a-PaAACT. Result: The open reading frame (ORF) of PaAACT gene was 1 254 bp,and encoded 417 amino acid residues. Bioinformatic analysis showed that the molecular formula of PaAACT protein was C1 914H3 120N538O576S17,inferring that its molecular weight was 43.43 kDa,the theoretical isoelectric point was 8.90,and the instability index of PaAACT protein was 32.27,which was a stable protein. According to bioinformatics analysis,PaAACT protein was a member of the thiolase family and contained one conserved site and one active site of the thiolase family at the C-terminal. PaAACT protein may be located in the cytoplasm,without a signal peptide or transmembrane domain. The phylogenetic analysis indicated that PaAACT protein showed the highest homology with AACT protein from polygonaceae plants (such as Beta vulgaris). The recombinant PaAACT protein was successfully expressed in Escherichia coli BL21(DE3) strain through IPTG induction, and the purified target protein was obtained by Ni2+ affinity chromatography. Conclusion: In this study,the PaAACT gene was cloned from P. americana,which lays a foundation for further determination of enzyme activity assay of PaAACT and preparation of antibody,and provides the theoretical basis for studying its role in the biosynthesis pathway of EsA.
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In order to study the biosynthesis pathway of esculentoside A, the Illumina HiSeq 4000 highthroughput sequencing method was used to analyze the transcriptome of Phytolacca americana seedlings. The 9.60 Gb clean data were obtained after the transcriptome of P. americana assembled by Trinity software. The total 63 957 unigenes were obtained after assembly and the average length was 988.82 bp, among them 24 517 unigenes (38.33%) were annotated in the public databases Nr, Swiss-Prot, COG, KOG, Pfam, GO and KEGG. According to the assignment of KEGG pathway, 53 unigenes were involved in terpenoid backbone biosynthesis and 8 unigenes involved in triterpenoid biosynthesis. Additionally, there were 417 unigenes assigned to other secondary metabolic pathways in P. americana. The post-modification enzyme genes involved in the esculentoside A biosynthesis were also analyzed in the transcriptome of P. americana. The results indicated that 130 unigenes may have the function of CYP450 which was involved in oxidation/hydroxylation modification of P. americana secondary metabolites. Furthermore, 46 unigenes had the function of glycosyltransferase UGT. The transcriptome data of P. americana laid a foundation for studying the biosynthesis pathway of esculentoside A and other secondary metabolites, and also provided theoretical basis for formation of medicinal materials quality.
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In order to explore genetic basis for the biosynthesis of secondary metabolism,the transcriptome of Cornus officinalis was sequenced by the new generation of high-throughput sequencing technology,A total of 96 032 unigenes were assembled with an average length of 590.53 bp. Among them, 35 478 unigenes were annotated in the public databases NR,Swissprot,COG,GO,KOG,Pfam and KEGG. Based on the assignment of KEGG pathway, 84 involved in ridoid biosynthesis and 487 unigenes involved in others secondary metabolites biosynthesis were found. Additionally,53 unigenes and 72 unigenes were predicted to have potential functions of cytochome P450 and UDP- glycosyltransferases based on the annotation result, which may encode responsible for secondary metabolites modification. This study was the first comprehensive transcriptome analysis for C. officinalis, and the candidate genes involved in the biosynthesis of secondary metabolites were obtained. The transcriptome data constitutes a much more abundant genetic resource that can be utilized to benefit further molecular biology studies on C. officinalis.