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Objective To clone cDNA sequence of geranyl pyrophosphate synthase (GPS) gene from Eleutherococcus senticosus and analyze genetic characteristics, gene expression level in different organs and the correlation between GPS gene expression and saponins content. Methods RNA was extracted from E. senticosus and reverse transcribed into cDNA. Gene specific primers were designed according to the unigene (c37362.graph_c0) of GPS from transcriptome sequencing data. The full length of the GPS gene cDNA was amplified by PCR. The expression level of GPS gene in different organs was analyzed by qRT-PCR. The content of E. senticosus saponins was detected by spectrophotometry method. Results GPS gene cDNA was cloned from E. senticosus and encodes 419 amino acids with full length of 1 260 bp. GPS protein located in mitochondria does not have transmembrane region. The GPS gene was expressed in each organ and had the highest expression in blade, which is 5.26 times in root. The relative expression of GPS gene and content of saponin showed the same trend and significant positive correlation (r = 0.851, P < 0.05). Conclusion The whole length of cDNA sequence of GPS gene is cloned for the first time, and there is a positive correlation between the expression level of GPS gene and the saponin content of E. senticosus.
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Objective: To clone and analyze the full length DNA and promoter sequence of GAPDH gene from Eleutherococcus senticosus. Methods: PCR and TAIL-PCR techniques were used to clone the full length DNA sequence and promoter sequence of GAPDH gene of E. senticosus, then these two sequences were analyzed by bioinformatics methods. Results: Length of 4 103 bp of E. senticosus GAPDH gene DNA and promoter sequence was cloned. Gene structure analysis showed that it contained 12 exons and 11 introns, and the splicing principles of its exon and intron were consistent with GT-AG; The promoter sequence length was 1 304 bp, and the transcription start site located 61 bp upstream of the initiation codon ATG; The promoter elements such as TATA-box, CAAT-box, as well as many cis-regulatory elements were related to hormone signal response, light response and stress signals. Conclusion: The full length DNA and promoter sequence of GAPDH gene in E. senticosus is successfully cloned and reported for the first time, and it provides a stable foundation for further study of GAPDH gene structure and function of E. senticosus.
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Objective: To obtain the transcriptome database and differentially expressed genes of Eleutherococcus senticosus. Methods: We choose the high content group and the low content group of saponin as experimental materials, and use the high-throughput sequencing technology (Illumina HiSeq 4000) to sequence the transcriptome of E. senticosus, then we systematically analyze the sequencing results in the bioinformatic way. Results: We have assembled 8.34 Gb database, after assembly steps, we get 77 087 of E. senticosus unigenes, then blasting them with five data banks. All unigenes are involved in 55 GO-terms and 116 metabolic pathways. Though the differentially expressed analysis of two materials, we get 530 differentially expressed genes, the up-regulated genes account for 42.08%, the down-regulated genes account for 57.92%. After GO and Pathway enrichment analysis, we get 408 GO-natations and 40 metabolic pathways. Conclusion: These data represent the abundant messeges about transcripts and provide the valuable genome data sources in molecular biology of E. senticosus.
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Objective: To clone β-amyrin synthase (bAS) gene from Eleutherococcus senticosus and analyze the effect of its expression on saponin contents. Methods: The sequence of cDNA of E. senticosus bAS was cloned by homologous cloning strategy. Quantitative real time PCR was developed to analyze the expression pattern of E. senticosus bAS gene. And E. senticosus saponin contents were measured by spectrophotometry method. Results: Length 1 223 and 1 226 bp of E. senticosus bAS1 and bAS2 genes were cloned. The results showed that bAS1 and bAS2 were expressed in the each growth period and every organ of E. senticosus, and the expression differed significantly (P < 0.05). bAS1 showed the highest expression when the plants were grown in germination stage, then rapidly depressed, and changed slightly in the end. The expression of bAS2 showed the characteristic of low-high-low. The expression of bAS1 in different organs of E. senticosus was constant, but the highest content of the expression of bAS2 was in the leaves. With the treatment of methyl jasmonate (MeJA), bAS2 expression has been significantly improved and bAS1 without a significant changing. There exists significantly positive correlation (P < 0.01) between the content of E. senticosus saponins and the expression levels of bAS2, and bAS1 without a significant difference. Conclusion: bAS2 may be a key enzyme gene in the biosynthesis of triterpenoid saponins.
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Objective: To analyze the codon usage of functional genes in Eleutherococcus senticosus and their influencing factors. Methods: The multivariate statistical analysis and correspondence analysis were carried out using CodonW and SPSS software with codon of 17 genes selected from the functional genes of E. senticosus. Results: GC contents at the three positions of functional gene codons in E. senticosus was 51.03%, 41.23%, and 40.04%, all of which had a significant correlation coefficient (P < 0.05). The correlation coefficients with GC12 and GC3 were 0.262, and both were insignificant. The relative synonymous codon usage of 27 codons was greater than 1, among which 22 codons ended with A or T base. In the corresponding analysis, the first axis showed the variation of 22.78%, and there were significant correlation coefficients in codon adaptation, codon bias index, and GC3 (P < 0.01). The correlation coefficients were 0.686, 0.617, and 0.786, respectively, but they were insignificantly related with the effective number of codons (ENC). The second axis showed the variation of 19.28% and it was only significantly related with ENC (r = 0.635). Seventeen optimized codons in functional genes of E. senticosus were defined. Conclusion: All codons of functional genes in E. senticosus prefer to ending with A or T base. The codon usage bias is formed under the effects of mutation and selection.
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Objective: To clone the actin (ACT) gene of Eleutherococcus senticosus, and to make the gene a valuable internal gene. Methods: Part of the sequence of ACT gene was cloned by real-time PCR (RT-PCR), and the sequence was used as internal control gene for analyses of semiquantitative PCR and RT-PCR. Results: The ACT gene (1031 bp) of E. senticosus was cloned, coding 343 amino acids. To compare the amino acid sequence of E. senticosus ACT gene with those of Betula luminifera, Gossypium hirsutum, and Camellia sinensis, the amino acid homology was 99.42%, 98.83%, and 98.54%. The expression of ACT in different organs of E. senticosus during various growing periods was constant. The expression of ACT gene in different organs and during different growth and development stages was basically constant, and when the sequence acted as internal control gene, the semiquantative PCR and RT-PCR have good amplification effect and reproducibility. Conclusion: The ACT sequence in E. senticosus is firstly separated and reported, it could act as an internal control gene, and its reaction system of RT-PCR is established.