Chinensis Cells Research Articles

TcMYB29a, an ABA-Responsive R2R3-MYB Transcriptional Factor, Upregulates Taxol Biosynthesis in Taxus chinensis.

Paclitaxel (Taxol), a highly modified diterpene agent mainly obtained from Taxus species, is the most widely used anticancer drug. Abscisic acid (ABA) is a well-known stress hormone that plays important roles in the secondary metabolism of plants, and it can also induce the accumulation of taxol in Taxus cell suspension cultures. However, the mechanism behind the regulation of taxol biosynthesis by ABA remains largely unknown. In previous research, a R2R3 MYB transcription factor (TF) TcMYB29a was observed to show a significant correlation with taxol biosynthesis, indicative of its potential role in the taxol biosynthesis. In this study, the TcMYB29a encoded by its gene was further characterized. An expression pattern analysis revealed that TcMYB29a was highly expressed in the needles and roots. Overexpression of TcMYB29a in Taxus chinensis cell suspension cultures led to an increased accumulation of taxol, and upregulated expression of taxol-biosynthesis-related genes, including the taxadiene synthase (TS) gene, the taxane 5α-hydroxylase (T5OH) gene, and the 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT) gene as compared to the controls. Chromatin immunoprecipitation (ChIP) assays, yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs), and dual-luciferase reporter assays verified that TcMYB29a could bind and activate the promoter of TcT5OH. Promoter sequence analysis of TcMYB29a revealed that its promoter containing an AERB site from -313 to -319 was a crucial ABA-responsive element. Subsequently, the ABA treatment assay showed that TcMYB29a was strongly upregulated at 6 h after ABA pretreatment. Furthermore, TcMYB29a was strongly suppressed at 3 h after the methyl jasmonate (MeJA) treatment and was depressed to the platform at 12 h. Taken together, these results reveal that TcMYB29a is an activator that improves the accumulation of taxol in Taxus chinensis cells through an ABA-medicated signaling pathway which is different from JA-medicated signaling pathways for the accumulation of taxol. These findings provide new insights into the potential regulatory roles of MYBs on the expression of taxol biosynthetic genes in Taxus.

A phenazine-imidazole based ratiometric fluorescent probe for Cd2+ ions and its application in in vivo imaging.

A new phenazine-imidazole based Schiff base (PIS) fluorescent probe has been developed for the ratiometric detection of Cd2+ ions in aqueous media at physiological pH. PIS upon binding with Cd2+ ions shows red shifted fluorescence and thereby, permits ratiometric detection of Cd(II) ions. A detection limit down to 2.10 × 10-8 M was determined for Cd2+ quantitation. Also, the accompanying apparent fluorescence color change (from yellow to orange red) is noticeable to the naked eye under a UV-lamp. The sensing mechanism could be attributed to the 1 : 1 PIS-Cd complexation, followed by extension of the conjugation due to better planarity and modulation of the charge transfer efficiency in the probe. This was complemented by solvatochromism and density functional theory calculations. Furthermore, PIS was used to detect Cd2+ in Oxya chinensis cells, zebrafish larvae and live tissues of Arabidopsis thaliana under a fluorescence microscope, showing great potential in analyzing living biosystems.

Kinetics, Mechanism, and Thermodynamics Studies of Vacuum Drying of Biomass from Taxus chinensis Cell Cultures

In this study, we investigated the kinetics, mechanism, and thermodynamics of vacuum drying and dehydration of plant cells of Taxus chinensis. The efficiency of vacuum drying increased with increases in the drying temperature (45–60°C). When the experimental data were fitted to typical kinetic models for drying, the Modified Page were the most appropriate. Thermodynamic parameters revealed the endothermic, irreversible, and spontaneous nature of the drying. The effective diffusion coefficient (3.6602 × 10−14−10.9810 × 10−14 m2/s) and mass transfer coefficient (2.2536 × 10−11−10.8110 × 10−11 m/s) increased with increasing drying temperature. The small Biot number (2.6167 × 10−2−4.1841 × 10−2) indicated that the process of mass transfer was externally controlled.

Adsorption Kinetics, Mechanism, Isotherm, and Thermodynamic Analysis of Paclitaxel from Extracts of Taxus chinensis Cell Cultures onto Sylopute

The adsorption characteristics of paclitaxel from extracts of Taxus chinensis cell cultures by the commercial adsorbent Sylopute were investigated using different parameters such as initial paclitaxel concentration, adsorption temperature, and contact time. The equilibrium adsorption data were best represented with the highest accuracy (r2 > 0.984, RMSD<0.0009) by the Langmuir isotherm. It was found that the adsorption capacity increased with increasing temperatures and the adsorption of paclitaxel onto Sylopute was favorable. The kinetic data fit the pseudo-second-order model well, and intraparticle diffusion played a dominant role in paclitaxel adsorption according to the intraparticle diffusion model. Thermodynamic parameters such as ΔH0, ΔS0, and ΔG0 were also calculated. The results indicate that the process of paclitaxel adsorption onto Sylopute is endothermic and nonspontaneous. The isosteric heat of adsorption, ΔHx (20–25 kJ/mol), was nearly a constant, even with variations in surface loading (qe: 3–9 mg/g), indicating a homogeneous surface.

Biodiesel Production: Utilization of Loofah Sponge to Immobilize Rhizopus chinensis CGMCC #3.0232 Cells as a Whole-Cell Biocatalyst.

Rhizopus chinensis cells immobilized on loofah (Luffa cylindrica) sponges were used to produce biodiesel via the transesterification of soybean oil. In whole-cell immobilization, loofah sponge is considered to be a superior alternative to conventional biomass carriers because of its biodegradable and renewable properties. During cell cultivation, Rhizopus chinensis mycelia can spontaneously and firmly adhere to the surface of loofah sponge particles. The optimal conditions for processing 9.65 g soybean oil at 40°C and 180 rpm using a 3:1 methanol-to-oil molar ratio were found to be 8% cell addition and 3-10% water content (depending on the oil's weight). Under optimal conditions, an over 90% methyl ester yield was achieved after the first reaction batch. The operational stability of immobilized Rhizopus chinensis cells was assayed utilizing a 1:1 methanol-to-oil molar ratio, thus resulting in a 16.5-fold increase in half-life when compared with immobilized cells of the widely studied Rhizopus oryzae. These results suggest that transesterification of vegetable oil using Rhizopus chinensis whole cells immobilized onto loofah sponge is an effective approach for biodiesel production.

A systematic approach to expound the variations in taxane production under different dissolved oxygen conditions in Taxus chinensis cells.

Our results provide an evidence that the changes in taxane production caused by dissolved oxygen shifts could be associated with the global variations in the cell central carbon metabolism. Taxol is an important taxane synthesized by the Taxus plant. A two-stage culture of Taxus in vitro has been considered as an attractive alternative approach to produce Taxol and its precursors. To investigate the consequences of dissolved oxygen (DO) shifts for cell primary and secondary metabolism, we conducted metabolomic and transcriptomic profiling analyses under low dissolved oxygen (LDO), medium dissolved oxygen (MDO), and high dissolved oxygen (HDO) conditions in a suspension culture of Taxus chinensis cells. Under LDO, the results indicate a significant increase in the production of Taxol and its main precursors by 3.4- to 1.4-fold compared with those under MDO and HDO on 9th day. Multiple acyl taxanes (MAT) are abundant taxanes in the cells, and exhibited only a slight increase under the same conditions. Metabolomic analysis based on 209 primary metabolites indicated that several pathways in central carbon metabolism were involved, including the enhancement of the glycolysis pathway of glucose-6-phosphate to fructose-6-phosphate and pyruvate and the mevalonate pathway of terpene biosynthesis, and decline in the tricarboxylic acid pathway under LDO. These results indicate the mechanism by which related taxanes accumulate through enhancing the supplies of substrates and expression levels of hydroxylases. Excess acetyl-CoA supply induced by high oxygen stress was found to be correlated with high productivity of MAT. Our results provide an evidence that the changes in taxane production caused by DO shifts could be associated with the global variations in the cell central carbon metabolism.

Mechanisms and effective control of physiological browning phenomena in plant cell cultures.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Enhancing taxane biosynthesis in cell suspension culture of Taxus chinensis by overexpressing the neutral/alkaline invertase gene

Sucrose metabolism generates material and energy foundations and regulates the biosynthesis of secondary metabolites in plant cell cultures. However, this regulation mechanism is unclear. The neutral/alkaline invertase (NINV) gene is among the key enzymes in sucrose hydrolysis. Thus, the complete open reading frame (ORF) of this gene was obtained from Taxus chinensis cells. This gene was also successfully expressed in Escherichia coli. The expressed protein was verified through the function of sucrose cleavage. Furthermore, the transcriptional levels of the NINV gene can respond to the sucrose concentration changes and relate to taxane biosynthesis in T. chinensis cells. Subsequently, we developed the gene expression vector and introduced it into T. chinensis suspension cells to confirm the role of the NINV gene in taxane biosynthesis. Results indicated that this gene was overexpressed and that it significantly enhanced the expression of the taxadiene synthase (TAS) gene, as well as the biosynthesis of the seven detected individual taxanes. Thus, NINV-mediated sucrose metabolism can promote the biosynthesis of secondary metabolites. This study presents new concepts and approaches to increase the production of secondary metabolites in plant cell cultures.

Application of High-Speed Counter-Current Chromatography for Isolation and Purification of Paclitaxel and Related Taxanes from Taxus chinensis Cell Culture

Six taxanes were isolated and purified from Taxus chinensis cell culture extract using high-speed counter-current chromatography. The crude cell culture extract was first treated with Al2O3 column chromatography and then divided into two parts: fraction 1 and fraction 2. 52 mg of taxuyunnanine C and 11 mg of sinenxane C were yielded from 150 mg of fraction 1. 5 mg of 9-dihydrobaccatin III, 12 mg of baccatin III, 12 mg of paclitaxel, and 17 mg baccatin VI were yielded from 150 mg of fraction 2. The purities of the six compounds were 93.51%, 98.02%, 98.61%, 97.55%, 98.93%, and 98.76%.

Preparative isolation of paclitaxel and related taxanes from cell cultures of Taxus chinensis using reversed-phase flash chromatography

In this study, paclitaxel, baccatin III, taxuyunnanine C and sinenxane C were successfully separated by reversed-phase flash chromatography on a manually packed C18 column from Taxus chinensis cell culture extract. The crude cell culture extract was first treated with Al2O3 column chromatography and then divided into two parts: fraction 1 and fraction 2. Ten milligrams of baccatin III and 19 mg of paclitaxel were obtained from 100 mg dried fraction 1. Fifty-two milligrams of taxuyunnanine C and 11 mg sinenxane C were obtained from 100 mg dried fraction 2. The purities of the four compounds were 98.02%, 98.53%, 98.93% and 98.76%, respectively. Their structures were characterised by using UV, MS and NMR. These results indicate that paclitaxel and related taxanes including baccatin III can be obtained from cell culture in a highly pure state using reversed-phase flash chromatography.

Correlation analysis of the taxane core functional group modification, enzyme expression, and metabolite accumulation profiles under methyl jasmonate treatment

Metabolomic and transcriptomic profiling data were obtained and integrated to elucidate the crucial network controls on taxol and its precursor biosynthesis during the taxane core functionalization within methyl jasmonate (MJ)-induced Taxus chinensis cells. Twelve metabolites were identified using liquid chromatography-electrospray ionization-mass spectrometry. These metabolites contain taxol (paclitaxel), baccatin III (B-III) and its analogs, a group structurally bearing multiple free hydroxyls (TAX), and another group of multiple acyl taxanes (MAT), including taxuyunnanine C (TC) and its analogs. The metabolomic profile showed a higher increase in TAX than in MAT. Particularly, the ratio of B-III and taxol to the total taxane content increased more significantly in TAX than in MAT. The MAT proportion did not significantly change, although they are predominant components in cell cultures compared with TAX. Quantitative real-time polymerase chain reaction (qRT-RCR) was used to determine the transcription level of 20 genes, among which 11 were reported responsible for taxol biosynthesis and 9 were obtained from our previous transcriptomic data. The total expression levels of hydroxylase after 24 h and 6 days were higher than those of acylase. The principal component analysis (PCA) results validated the metabolomic analysis data, indicating that hydroxylation was more crucial than acylation for controlling the flux toward TAX biosynthesis. Furthermore, the PCA contribution comparison showed that two undefined genes of OHX1 and ACX3 might have good potential in TAX upregulation and MAT downregulation. To the best of our knowledge, this study provides the first experimental evidence on the contribution of total hydroxylation to taxane biosynthesis.

Improved assessment of aggregate size in Taxus plant cell suspension cultures using laser diffraction

In suspended culture, most relevant for biotechnological application, plant cells form aggregates. This phenomenon is of importance as it is related to productivity, leads to local heterogeneities, and might be a reason for the considerable shear sensitivity of these cultures. The valid measurement of plant cell aggregates, however, is not trivial, due to a rather large size distribution and measurement artifacts implied by the measuring method. In this study, laser diffraction was used as a novel method for characterization of Taxus chinensis cells, a major source for the antitumor agent paclitaxel. Aggregate size measured in shaking flask cultivations over 10 days revealed an increase during the growth phase of a batch cycle and a decrease during the stationary phase. During growth, the increase in bio dry weight was proportional to aggregate size. Laser diffraction was found superior to microscopy and image analysis, which had a tendency to underestimate aggregate size up to 20%. This novel approach provides a practicable, rapid, robust, and reproducible way to analyze a 100-fold more samples in considerably less time than image analysis and is therefore of especial value for quality control in industrial plant cell cultivation.

Transcriptional profile of Taxus chinensis cells in response to methyl jasmonate

BackgroundMethyl jasmonate (MeJA) has been successfully used as an effective elicitor to enhance production of taxol and other taxanes in cultured Taxus cells. However the mechanism of MeJA-mediated taxane biosynthesis remains unclear. Genomic information for species in the genus Taxus is currently unavailable. Therefore, information about the transcriptome of Taxus cells and specifically, description of changes in gene expression in response to MeJA, is needed for the better exploration of the biological mechanisms of MeJA-mediated taxane biosynthesis.ResultsIn this research, the transcriptome profiles of T. chinensis cells at 16 hours (T16) after MeJA treatment and of mock-treated cells (T0) were analyzed by “RNA-seq” to investigate the transcriptional alterations of Taxus cell in response to MeJA elicitation. More than 58 million reads (200 bp in length) of cDNA from both samples were generated, and 46,581 unigenes were found. There were 13,469 genes found to be expressed differentially between the two timepoints, including all of the known jasmonate (JA) biosynthesis/JA signaling pathway genes and taxol-related genes. The qRT-PCR results showed that the expression profiles of 12 randomly selected DEGs and 10 taxol biosynthesis genes were found to be consistent with the RNA-Seq data. MeJA appeared to stimulate a large number of genes involved in several relevant functional categories, such as plant hormone biosynthesis and phenylpropanoid biosynthesis. Additionally, many genes encoding transcription factors were shown to respond to MeJA elicitation.ConclusionsThe results of a transcriptome analysis suggest that exogenous application of MeJA could induce JA biosynthesis/JA signaling pathway/defence responses, activate a series of transcription factors, as well as increase expression of genes in the terpenoid biosynthesis pathway responsible for taxol synthesis. This comprehensive description of gene expression information could greatly facilitate our understanding of the molecular mechanisms of MeJA-mediated taxane biosynthesis in Taxus cells.

Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T.chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T.chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T.chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T.chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T.chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures.

Enhancing Taxol Biosynthesis by Overexpressing a 9-Cis-Epoxycarotenoid Dioxygenase Gene in Transgenic Cell Lines of Taxus chinensis

Plant secondary metabolites constitute are a wide range of compounds whose biosynthesis takes place in response to biotic and abiotic stresses. The phytohormone abscisic acid (ABA) acts as an important signaling molecule that regulates plant response to various stresses. Moreover, 9-cis-epoxycarotenoid dioxygenase (NCED) is one of the key enzymes in the ABA biosynthesis pathway in higher plants. In this study, a new NCED gene from Taxus chinensis, the TcNCED1, was overexpressed in transgenic T. chinensis cells, resulting in a maximum of 48 % more accumulation of ABA and a 2.7-fold increase of taxol production compared to the untransformed cells, respectively. These results indicate that overexpression of TcNCED1 can significantly increase the ABA and taxol level in T. chinensis cells, which probably provides an alternative approach in metabolic engineering to improve the yield of taxol in T. chinensis cells through genetic manipulation of the related genes in the ABA biosynthetic pathway.

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone-induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone-triggered ABA generation and assayed the effect of fluridone on ozone-induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone-triggered ABA generation but also blocked the ozone-induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose-dependent. Interestingly, the suppression of fluridone on ozone-induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone-induced Taxol production of T. chinensis suspension cell cultures.

Intracellular salicylic acid is involved in signal cascade regulating low ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis

It was previously reported that low initial ammonium (2 mM) in medium had significant stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells. However, the secondary metabolism induction mechanism of the low initial ammonium is yet unknown in plant cells. To provide an insight into the defense signals response to the low initial ammonium, oxidative burst and intracellular salicylic acid (SA) were detected, and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in the cell cultures of T. chinensis. Induced H(2)O(2) production, elevated phenylalanine ammonia-lyase (PAL) activity, and enhanced SA biosynthesis were observed. Interestingly, inhibition of SA biosynthesis by paclobutrazol and (BOC-aminooxy) acetic acid significantly depressed the Tc stimulation and up-regulation of Tc biosynthetic genes of geranylgeranyl diphosphate synthase and taxadiene synthase. The role of intracellular SA in regulating Tc biosynthesis was further confirmed by applying exogenous SA in normal ammonium (20 mM) medium. The results indicated that SA acted as a signal in low initial ammonium-induced Tc biosynthesis. A signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

Overexpression of a 10-deacetylbaccatin III-10 β-O-acetyltransferase gene leads to increased taxol yield in cells of Taxus chinensis

Taxus chinensis suspension cells were transformed by the Agrobacterium-mediated transformation method to overexpress the dbat gene coding for 10-deacetylbaccatin III-10 β-O-acetyltransferase, a key enzyme for taxol biosynthesis. A. tumefaciens strain LBA4404 harboring either pCAMBIA1303 or the recombinant plasmid p1303-SdbatN was used. Both plasmids harbored the hygromycin phosphotransferase gene (hptII) and gusA-mgfp5 gene as selectable markers, but the latter plasmid also harbored the dbat gene in the T-DNA region. The transgenic T. chinensis cells had been maintained in modified Gamborg’s B5 medium supplemented with hygromycin for more than 14 months and were subcultured at 4-week intervals. The selected transgenic cells were identified by PCR, Southern blot analysis, β-glucuronidase assay and western blot analysis, and the results showed that the transgenes were integrated in the chromosomal DNA of T. chinensis cells with single-copy style, and that the gusA-mgfp5 reporter gene was expressed in the transgenic cells. The dbat mRNA expression level in the p1303-SdbatN-transgenic T. chinensis cells tested by real-time quantitative PCR was 5.3 ± 0.6 times that of the non-transformed cells. Taxol yield of the p1303-SdbatN-transgenic T. chinensis cells was about 1.7 times that of the non-transformed cells. These results suggest that the overexpression of dbat gene in transgenic T. chinensis cells leads to increased taxol yield.

Production of two intermediate taxoids, 2-hydroxy- 5α,10β-diacetoxytaxadiene and 2-hydroxy-5α,10β,14β- triacetoxytaxadiene, from Taxus chinensis cell culture

The production of taxuyunnanine C (Tc) (2α,5α,10β,14β-tetraacetoxytaxa-4(20),11(12)-diene) was significantly enhanced when Taxus chinensis cell cultures were treated with methyl jasmonate (MJA) or 2,3-dihydroxypropyl jasmonate (DHPJA), a newly synthesized jasmonate analog, in combination with in situ absorption. Along with the production of Tc, two intermediate taxoids were produced and purified. On the basis of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectral data, the chemical structures of the two intermediate taxoids were found to be 2-hydroxy-5α,10β-diacetoxytaxadiene and 2-hydroxy-5α,10β,14β- triacetoxytaxadiene. This is the first study to report the production of these two taxoids using plant cell culture. Key words : Taxus chinensis , taxuyunnanine C, methyl jasmonate, 2,3-dihydroxypropyl jasmonate, in situ absorption, 2-hydroxy-5α,10β-diacetoxytaxadiene, 2-hydroxy-5α,10β,14β-triacetoxytaxadiene.

Effect of DNA Methylation on 18S rRNA Gene Sequences during Culture of Taxus chinensis Cells

* Author for correspondence and reprint requests Z. Naturforsch. 64c, 418-420 (2009); received December 15, 2008 Cell suspension culture has rapidly become an alternative source of taxol, an anticancer compound. To investigate the role of DNA methylation in the cultural course of Taxus chinensis cells, analyses of 18S rRNA gene sequences of cultured T chinensis cells and related species were conducted. The phylogenetic analysis of 18S rRNA gene sequences indicated that HG-1 (the cultured T chinensis cells), like T mairei (the natural variety of T chinensis), should be a new variety of T chinensis, and cell culture can change the 18S rRNA gene sequence at the level of species despite 18S rRNA is the most conserved gene. The analyses of the CpG and TpG+CpA relative abundance and GC content of the 18S rRNA gene sequences made clear that DNA methylation contributed to changes of the 18S rRNA gene sequence of HG-1 at the level of species, which can make HG-1 to become a new variety of 7 chinensis.