高明波，張衛(wèi)，李興泰，阮成江，范圣第

1 生物化學工程國家民委-教育部重點實驗室，大連 116600

2 大連民族學院生命科學學院，大連 116600

3 澳大利亞Flinders大學 海洋分子生物過程及生物產(chǎn)品實驗室，阿德萊德 SA5042

中國紅豆杉細胞經(jīng)重復誘導和蔗糖飼喂后云南紫杉烷C生產(chǎn)的相應基因表達變化

高明波1,2，張衛(wèi)3，李興泰2，阮成江1,2，范圣第1

1 生物化學工程國家民委-教育部重點實驗室，大連 116600

2 大連民族學院生命科學學院，大連 116600

3 澳大利亞Flinders大學 海洋分子生物過程及生物產(chǎn)品實驗室，阿德萊德 SA5042

紅豆杉懸浮培養(yǎng)細胞具有可持續(xù)生產(chǎn)抗癌藥物紫杉醇及其他紫杉烷的潛力。在中國紅豆杉懸浮培養(yǎng)細胞中，云南紫杉烷 C(Tc) 是主要的次生代謝產(chǎn)物。為促使代謝前體由生成其他紫杉烷的代謝支路轉(zhuǎn)到生產(chǎn)紫杉醇，實驗采用實時定量PCR技術 (RQ-PCR) 揭示細胞培養(yǎng)過程中紫杉醇及紫杉烷合成關鍵基因的動態(tài)變化。在細胞培養(yǎng)的第7天和第12天，以100 μmol/L 2,3-二羥丙基茉莉酸 (DHPJA) 進行誘導，同時在第7天飼喂20 g/L的蔗糖，在此過程中考察6個關鍵基因 (TASY，TDAT，T5αH，TαH，T10βH和T14βH) 的表達變化。上述聯(lián)合調(diào)控手段使得Tc產(chǎn)量在第1次誘導8 d后達 (554.46±21.28) mg/L，第2次誘導9 d后高達 (997.72±1.51) mg/L。代謝早期基因TASY和TDAT在第1次誘導后表達量分別提高了182和98倍，在第2次誘導后表達量分別提高了208和131倍。在每次誘導后基因表達量提高約持續(xù)24 h，之后下降。其他4個基因 (T5αH、TαH、T10βH和T14βH) 的情況有所不同?；騎αH在2次誘導后表達量分別提高了3 061和1 016倍。其他3個基因T5αH、T10βH、T14βH在第1次誘導后表達量分別提高13、38、20倍，在第2次誘導后分別提高7、16、6倍。RQ-PCR結(jié)果表明基因表達和Tc積累之間存在緊密相關性：基因表達的變化與Tc產(chǎn)量的變化相一致，誘導可提高6個基因的表達量。基因的高表達隨著培養(yǎng)過程逐漸衰減，再次誘導可再次促使基因的高表達。

中國紅豆杉，云南紫杉烷C，2,3-二羥丙基茉莉酸，實時定量PCR

Abstract:Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol?)and other taxoids. In the cell culture of Taxus chinensis, Taxuyunnanine C (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, we employed Real-time Quantitative PCR(RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes (TASY, TDAT, T5αH, TαH, T10βH and T14βH) were quantified under the process condition of double elicitation by 2,3-dihydroxylpropanyl jasmonate (DHPJA) (100 μmol/L on day 7 and day 12), and sucrose feeding (20 g/L) on day 7. This process treatment led to a high accumulation of Tc at (554.46±21.28) mg/L 8 days after the first elicitation. Then 9 days after the second elicitation, Tc production was as high as (997.72±1.51) mg/L. The early pathway genes TASY and TDAT were significantly up-regulated by 182-fold and 98-fold, respectively for the first DHPJA elicitation and by 208-fold and 131-fold,respectively for the second elicitation. The induction occurred after each elicitation lasted for about 24 h before their abundances decreased. Things are somewhat different in the case of the other four genes T5αH, TαH, T10βH and T14βH. For gene TαH, it was highly up-regulated by 3061-fold for the first DHPJA elicitation and by 1016-fold for the second elicitation. For the other three genes T5αH, T10βH, T14βH, they were up-regulated by 13-fold, 38-fold and 20-fold, respectively for the first DHPJA elicitation and by 7-fold, 16-fold and 6-fold, respectively for the second elicitation. The RQ-PCR results showed that there is tight correlation between gene expression and Tc accumulation. Gene expression was in accordance with Tc yield. Elicitation could improve expression of six genes. While along with culture course, high expression of the genes weakened. Elicitation for the second time would promote high expression of the genes again.

Keywords:Taxus chinensis, Taxuyunnanine C, 2,3-dihydroxylpropanyl jasmonate, Real-time Quantitative PCR

Paclitaxel (Taxol?, Bristol-Myers Squibb), a famous anti-cancer drug, is one side-chain taxane, which is structurally more complex and representative of more than 350 taxoids[1-2]. At present, semi-synthesis is the main source of paclitaxel: 10-deacetylbaccatin-III, a paclitaxel precursor that is harvested from Taxus needles, is converted to paclitaxel via chemical synthesis. Another source approved by FDA was through plant cell culture, which is environment friendly and is expected to contribute significantly for the sustainable supply of paclitaxel[3].

Introduction

A noticeable phenomenon is that considerable precursors fluw to taxoid rather than paclitaxel in both intact Taxus tissues[4]and derived cell cultures[5].Among the taxoids, 14β-hydroxy taxoids (Tc and its relatives) are most usual. C-14 hydroxylation is probably the early sideway of taxane synthesis. Taxol?has no oxygen substitute at C-14. Tc is a novel compound which has a neuron growth factor(NGF)-like activity[6].

A clear understanding of paclitaxel biosynthetic pathway regulation is necessary to develop a superior strain for the supply of paclitaxel or regulate (either up or down) the genes that encode Taxol?or taxoid pathway steps. Although the taxane biosynthetic pathway is not fully clarified, the first three steps of paclitaxel biosynthesis have been elucidated. Derived from isoprenyl diphophate (IPP) and dimethylallyl diphosphate (DAPP), geranylgeranyl pyrophosphate(GGPP) is synthesized by geranylgeranyl pyrophosphate synthase (GGPPS)[7]. GGPP is converted to taxa 4(5),11(12)-diene by taxadiene synthase (TASY) to establish the taxane ring[8-10]and then to taxa 4(20),11(12)-dien-5α-ol by taxadiene 5α hydroxylase(T5αH)[11]. The first hydroxylation at the C 5α-position, with allylic migration of the double bond occurred. A branch occurred at this point[12-13].Taxadiene 13α-hydroxylase (TαH) converts taxa 4(20),11(12)-dien-5α-ol to taxa 4(20), 11(12)-dien-5α,13αdiol[14]. The alternate branch in the pathway implements taxadiene 5α-ol O-acetyltransferase(TDAT) to form 4(20), 11(12)-dien-5α-yl acetate from 4(20), 11(12)-dien-5α-ol, which is then further converted to 4(20), 11(12)-dien-5α-acetoxy-10β-ol by taxane 10β hydroxylase (T10βH). Only taxanes withC-5 acetyl oxygen substitute and C-10 hydroxylation can be catalyzed by Taxane 14β-hydroxylase (T14βH)to form C-14 hydroxyl substitute. The cytochrome P450 taxoid 14β-hydroxylase, principally utilizes the C5-acetate esters of 5α-hydroxytaxadiene and 5α,10β-dihydroxytaxadiene as preferred substrates[15].The steps leading from the acetate or the diol intermediates to functionalized taxanes are unknown.

The last steps have been already characterized by direct cloning methods. 10-deacetylbaccatin III, the important semi-synthetic precursor of paclitaxel, is produced via taxane 2α-O-benzoyltransferase (DBBT).Then 10-DAB is converted to baccatin-III by 10-deacetylbaccatin-III-10-O-acetyltransferase (DBAT).Baccatin III: 3-amino, 3-phenylpropanoyltransferase(BAPT) ligates phenylisoserine (derived from phenylalanine via phenylalanine amin-omutase (PAM))to baccatin III to produce 3’-N-debenzoyl-2-deoxytaxol.3’-N-debenzoyl-2-deoxytaxol-N-benzoyltransferse(DBTNBT) ligates a benzoyl CoA group to 3’-N-debenzoyl-2-deoxytaxol to produce 2’-deoxytaxol.Finally, benzamidation of 2’-deoxytaxol yields paclitaxel[16-19].

Methyl jasmonate (MJA) is commonly used to stimulate paclitaxel or taxane accumulation in Taxus plant cell culture. 2,3-dihydroxylpropanyl jasmonate(DHPJA), a MJA derivative synthesized by Qian[20],has been proved to have the ability to greatly enhance Tc accumulation[21].

In this paper, we examined the regulation of Tc biosynthetic pathway using a DHPJA responsive,well-characterized T. chinensis cell line. DHPJA was used twice to induce Tc synthesis in combination with sucrose feeding. The expression of 6 known pathway genes was examined by RQ-PCR. Tc was quantified via HPLC. We found that unelicited cultures produce low levels of Tc while DHPJA-elicited cultures accumulate much more Tc. The regulation of taxane biosynthetic pathway is proved to occur at the level of mRNA and that there is a tight correlation between steady-state transcript abundance and respective taxane accumulation.

1 Materials and methods

1.1 Plant cell line and suspension subcultures

T. chinensis cell line was used for all experiments and obtained as a gift from Prof. Jianjiang Zhong of Shanghai Jiaotong University. Suspensions were subcultured every 2 weeks in Murashige and Skoog medium basal salts with 30 g/L sucrose, supplemented with 0.5 mg/L of 6-benzyladenine (6-BA), 0.2 mg/L of 2,4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg/L of naphthaleneacetic acid (NAA) and 100 mg/L of ascorbic acid. The pH was adjusted to 5.8 before autoclaving. Cultures were maintained in a 500-mL Erlenmeyer flask containing 100 mL medium and cultured on a rotary shaker at 110 r/min and kept at(25±1) °C in the dark.

1.2 Double elicitation and sucrose feeding

For shake-flask cultures, 2 g fresh cells were inoculated in a 100-mL Erlenmeyer flask containing 20 mL medium with the same culture conditions as that of subcultures. On day 7, DHPJA was added to the cultures in 1 μL of ethanol per mL of culture medium at 100 μmol/L after being sterilized by filtering through 0.22 μm polyvinylidenedifluoride(PVDF) syringe filters (Millipore). The same volume of ethanol was added to the control. Simultaneously twenty grams per liter sterile sucrose were feeded on day 7. On day 12, DHPJA was added for the second time at 100 μmol/L. The cells were collected on day 0,7, 7.5, 8, 9, 12, 12.5, 13, 15, 18, 21, 24 and 30 for analysis. All experiments were performed in triplicate and the data are expressed as the means of three samples with standard deviation.

1.3 Taxane extraction from culture samples and quantification via HPLC

Taxane extraction and determination by HPLC were done according to the methodology described by Zhang[22].

For the extraction of taxane, 100 mg of powdered dry cells were soaked in 4-mL methanol and dichloromethane (1:1, V/V), then ultrasonicated for 30 minutes for six times. After centrifugation at 4 000 r/min for 10 min, the extract was evaporated to dryness at 25 °C by a rotary evaporator. The residue was dissolved in 4 mL dichloromethane and 1 mL distilled water and extracted for four times. After sufficient mixing, the mixture was centrifuged at 4 000 r/min for 10 min. The organic phase (the bottom layer) was collected and evaporated to dryness at 25 °C by a rotary evaporator. The residue was dissolved in 1 mL chromatograph- pure methanol and filtered through a0.22 μm PVDF syringe filter (Millipore).

A volume of 10 μL was analyzed by reverse-phase HPLC, using a Hewlett-Packard series 1100 HPLC system (Agilent). An alkyl phenyl column (250 mm×4.6 mm, 5 μm) was used at 25 °C. The mobile phase consisted of acetonitrile and water (58:42, V/V), and the flow rate was 1 mL/min. Taxane was monitored at 227 nm by using authentic standards as the reference.

1.4 RNA extraction and cDNA synthesis

Cells were collected by filtration from the media at low pressure and stored at -80 °C in polypropylene tubes. RNA was extracted using TRIZOL?reagent(Invitrogen life technologies) according to their handbook instructions. RNA quality detection was via formaldehyde denaturation agarose gel electrophoresis.

1.5 Probe creation

Probes for RQ-PCR were created by amplifying gene fragments from T. chinensis genomic DNA.Primers were designed from previously cloned cDNAs in regions conserved amongst T. brevifolia and are listed in Table 1. Amplification of fragments via PCR was performed using PCR Thermal Cycler (TaKaRa,Japan).

1.6 RQ-PCR

Quantification of total RNA was performed with Rotor-Gene 3000 Realtime PCR (Corbett Research).PCR reagents were as follows: 2.5 mmol/L dNTP(dATP, dGTP, dCTP and dTTP, 2.5 mmol/L each)(HyTest Ltd); 10×PCR buffer (Promega); 25 mmol/L MgCl2(Promega); Taq polymerase (Promega); 100 bp DNA ladder (Tiangen Biotech Co., LTD, Beijing.);10 000× Sybergreen (Molecular Probes).

PCR reaction system consisted of 25 μL, conditions were set as follows: 40 PCR cycles (94 °C, 20 s;annealing temperature, 20 s; 72 °C, 30 s); 72 °C elongated 5 min.

RQ-PCR conditions of housekeeping gene GAPDH and the other six genes are listed in Table 2. In order to establish melting curve of PCR products, temperature was raised slowly from 72 °C to 99 °C (1 °C increment every 5 seconds) after the completion of amplification reactions.

Table 1 Primers used to amplify gene fragments from T. brevifolia

Table 2 RQ-PCR conditions of housekeeping gene GAPDH and six selected genes

2 Results

2.1 Time course of dry cells and Tc accumulation

Fig. 1 and Fig. 2 showed the time courses of dry cells and Tc accumulation. As to dry cell weight,before day 18, the dry cell weight of double elicitation and sugar feeding was lower than that of control.While after day 18, due to sugar feeding on day 7, the dry cell weight of double elicitation and sugar feeding was higher than that of control.

A high accumulation of Tc was obtained at(554.46±21.28) mg/L 8 days after the first elicitation.And 9 days after the second elicitation, Tc production was as high as (997.72±1.51) mg/L.

Fig. 1 Dry cell weight of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A). Control: day 7 20 μL EtOH+0.5 mL H2O, day 12 20 μL EtOH; A: day 7 100 μmol/L DHPJA+20 g/L sucrose, day 12 100 μmol/L DHPJA.

Fig. 2 Tc production of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

2.2 Time courses of mRNA accumulation

As shown in Fig. 3 and Fig. 5, the early pathway genes TASY and TDAT were significantly up-regulated by 182-fold and 98-fold, respectively for the first DHPJA elicitation and by 208-fold and 131-fold,respectively for the second elicitation. The induction after each elicitation lasted for about 24 h before their abundances decrease. The mRNA levels of TASY and TDAT increased upon DHPJA elicitation at day 7, by reaching maximal levels at 24 h, then declined till another elicitation. The second peak was reached 24 h after DHPJA second elicitation at day12, then declined sharply.

Fig. 3 TASY mRNA expression of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

In the case of the other four genes T5αH, TαH,T10βH and T14βH, things are somewhat different. For gene TαH, it was greatly up-regulated by 3 061-fold for the first DHPJA elicitation and by 1016-fold for the second elicitation, as shown in Fig. 7. The first and second expression peak was reached 12 h and 24 h after each elicitation respectively. For the other three genes T5αH, T10βH, T14βH, they were up-regulated by 13-fold, 38-fold and 20-fold, respectively for the first DHPJA elicitation and by 7-fold, 16-fold and 6-fold, respectively for the second elicitation, as shown in Fig. 4, Fig. 6 and Fig. 8, respectively.

3 Discussion

Fig. 4 T5αH mRNA expression of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

Fig. 5 TDAT mRNA expression of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

Fig. 6 T10βH mRNA expression of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

Fig. 7 TαH mRNA expression of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

Fig. 8 T14βH mRNA expression of T. chinensis cell suspension cultures under control and double DHPJA elicitation with sucrose feeding (A).

The RQ-PCR results showed that there is tight correlation between gene expression and Tc accumulation.Changes of gene expression were in accordance with Tc yield changes. Elicitation could improve expression of six genes. While along with culture course, high expression of the genes weakened. Elicitation for the second time would promote high expression of the genes again.

When T. chinensis cell culture was elicited with DHPJA, there is a preference towards one side of the taxane biosynthetic pathway branch—13α-hydroxylation which coincides with the research of Ezekiel’s[3].While 13α-hydroxylated paclitaxel did not accumulate in our cell culture, this is because on one hand, TαH expression level is too low in the uninduced cell line.On the other hand, paclitaxel could only be produced after multiple steps behind 13α-hydroxylation. While 14α-hydroxylated Tc accumulated to a greater extent when cell cultures were treated with double elicitationand sucrose feeding because Tc yield was relatively high in the uninduced cell line. Our research work suggest that future efforts to enhance paclitaxel accumulation in Taxus cell suspension cultures should first focus on improving the TαH expression more greatly.

Acknowledgements

We are grateful to Prof. Jianjiang Zhong for providing the cell line and Prof. Xuhong Qian for the novel chemically synthesized jasmonate-DHPJA.

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Expression profiling of genes involved in Taxuyunnanine C biosynthesis in cell suspension cultures of Taxus chinensis by repeated elicitation with a newly synthesized jasmonate and sucrose feeding

Mingbo Gao1,2, Wei Zhang3, Xingtai Li2, Chengjiang Ruan1,2, and Shengdi Fan1
1 Key Laboratory of Biochemical Engineering (State Ethnic Affairs Commission-Ministry of Education), Dalian 116600, China

2 College of Life Science, Dalian Nationalities University, Dalian 116600, China
3 Molecular Bioprocessing and Bioproducts Laboratory, Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide SA 5042, Australia

Received: May 13, 2010; Accepted: October 9, 2010

Supported by: Mega-projects of Science Research for R&D of New Drugs (No. 2009ZX09102-237).

Corresponding author: Wenjie Tan. Tel/Fax: +86-10-63552140; E-mail: tanwj28@yahoo.com*These authors contributed equally to this study.

新藥創(chuàng)制科技重大專項 (No. 2009ZX09102-237) 資助。