Application strategy of DNA methylation and bisultite sequencing in medicinal plants / 中草药

DNA methylation is one of the most widely epigenetics phenomena, methylated DNA can regulate the phenotype of plants without changing the nucleotide sequence. In higher plants, there are three methylated sites, CG, CHG, and CHH (H stands for A, C, or T). They often occur in symmetrical sequences CG and repetitive sequence, and methylation degrees and patterns are different among different species. The methylation of functional gene promoters can suppress the gene expression in medicinal plants, and then affect the accumulation of secondary metabolites, resulting in quality variations. Among many methods for the DNA methylation detection, bisulfite sequencing can identify the site and extent of DNA methylation at single nucleotide level, which is the gold standard for DNA methylation analysis. Through optimizing primers and improving technology, it can effectively reveal the DNA methylation status for medicinal plants and provide technical support and a new research direction for clarifying some problems such as quality variations in medicinal plants, which are not completely explained by classical genetics.

Analysis of transicriptomes and exploring flavonoid biosynthetic pathway genes in Lithocarpus polystachyus / 中国中药杂志

The sweet taste and health effect of Lithocarpus polystachyus are mainly related flavonoid. To obtain Lithocarpus transcriptome database and flavonoid biosynthesis-related genes, the RNA-Seq techology (Illumina HiSeq 4000) was used to sequence its transcriptome. Six Gb database was assembled after assembly steps, and 41 043 of L. polystachyus unigenes were obtained. With blasting them with 7 data banks, all unigenes were involved in 51 GO-terms and 237 metabolic pathways. And furthermore 28 genes of the flavonoid biosynthesis-related were found. After using the MicroSatallite, 18 161 SSR were obtained, the single-nucleotide-repeated was the richest at 7 346. These data represent abundant messages about transcripts and provide valuable genome data sources in molecular biology of L. polystachyus.

Establishment of prokaryotic expression and optimization ox expression conditions of Eleutherococcus senticosus P450 gene / 中国中药杂志

According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.

Expression profiles analysis of two member of squaleneepoxidase gene family from Eleutherococcus senticosus / 中国中药杂志

In order to find the characteristics of two members of gene family of squaleneexpoxidase (SE) , a quantitative real time PCR method was developed to analyze the expression of Eleutherococcus senticosus SE1 and SE2 gene from different growth periods and in different organs. The result indicated that all the expression of SE2 more than SE1 in the whole growth period and organs of E. senticosus. And in the whole growth period, expression of SE1 showed a low-high-low characteristic. Both expression of SE2 and growth period showed the same trend. The lowest content of the expression was in the roots. SE1 expression have been improved more than SE2 when treated with MeJA. The expression of E. senticosus SE1 and saponins content had significantly positive correlation (P < 0.05) and the correlation coefficients was 0. 858, while the correlation was not significant for SE2. That indicated that SE1 played a key enzyme gene in the biosynthesis of triterpenoidsaponins

Analysis on bias of functional gene codon of Eleutherococcus senticosus / 中草药

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.

Cloning and expression stability analysis of actin gene in Eleutherococcus senticosus / 中草药

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.

Analysis on codon usage of chloroplast genome of Eleutherococcus senticosus / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze the codon usage of chloroplast genome and the influencing factor in Eleutherococcus senticosus.</p><p><b>METHOD</b>Codon of 52 genes, which were selected from the chloroplast genome sequence of E. senticosus, was multivariate statistical and correspondence analyzed using CodonW and SPSS software.</p><p><b>RESULT</b>GC content at the three position of codons by turns was 46.46%, 38.26%, 29.88%, whereas GC1 and GC2 had a significant correlation coefficient (P < 0.01). The correlation coefficient with GC12, and GC3 was 0.205 and was not significant correlated. There were 30 codons which relative synonymous codon usage was greater than 1 and 29 codons end with A and T. In the corresponding analysis, the first axis shows 10.35% variation. And there was significant correlation coefficient between ENC and GC3. The correlation coefficients with GC3 and ENC were -0.288 and 0.353, respectively. We defined 16 codons from 16 amino acids as the major preference codons in chloroplast genome of E. senticosus.</p><p><b>CONCLUSION</b>The third positions for all codon are preferred to ending with A and T. The codon usage bias is formed under effect of mutation and selection, as well as other factors. But the selection will have a far greater impact than others.</p>

Cloning and sequence analyses of Eleutherococcus senticosus GAPDH / 中草药

Objective In order to make the gene a valuable internal control gene, GAPDH of Eleutherococcus senticosus was cloned and analyzed in sequence. Methods Total RNA of E. senticosus was extracted using improved isothiocyanate method. Part sequence of GAPDH was coined by RT-PCR, and the sequence was acted as internal control gene for semiquantitative PCR. Results Length 627 bp of E. senticosus GAPDH was cloned, speculating coding 209 amino acids. To compare the amino acid sequence of £. senticosus GAPDH with those of Panax notoginseng, P. ginseng, and Arabidopsis thaliana, the amino acid homology was 97%, 93%, and 93%, respectively, and the nucleotide homology was 94%, 86%, and 84%, respectively. When the sequence acted as internal control gene, the semiquantative PCR has benign amplification effect and good reproducibility. Conclusion The cDNA clone of E. senticosus GAPDH is first reported, the results prove that the sequence is able to be internal control gene for analysis on gene expression. This study could make a foundation for the key enzyme expression and regulate mechanism analysis in eleutheroside biosynthesis pathway.