Botanical Studies (2011) 52: 191-196.

PHYSIOLOGY

Effects of low light on terpenoid indole alkaloid accumulation and related biosynthetic pathway gene expression in leaves of Catharanthus roseus seedlings

Ying LIU¹'², Dong-Mei ZHAO^1,2, Yuan-Gang ZU¹'²'*, Zhong-Hua TANG^1,2, Zhong-Hua ZHANG^1,2, Yang JIANG^1,2, and Dian-Yi SHI³

¹Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, People's
Republic of China

²Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, People s Republic of China

³National Maize Improvement Center of China, China Agricultural University, Beijing 100193, People s Republic of China

(Received January 11, 2010; Accepted September 9, 2010)

ABSTRACT. Catharanthus roseus (L.) G. Don, an important medicinal plant, produces the powerful alkaloids vinblastine (VBL) and vincristine (VCR), which are known for their remarkable inhibitory activity against tumor cells. This paper is the result of a study on the effects of low light/UV light on the contents of chlorophyll and alkaloids, and on the expression of terpenoid indole alkaloids (TIAs) biosynthesis pathway genes in the leaves of aseptic C. roseus seedlings. A plastic film treatment first induced a slight increase and then a significant decrease (p<0.05) in the total chlorophyll content, and a lower chlorophyll a/b ratio. The amounts of vindoline (VIN) and catharanthine (CAT) followed the identical pattern under plastic film but the content of VBL, generated by its precursors VIN and CAT, increased gradually until reaching a maximum (0.028 mg g^-1±0.0051) on the 15th day of treatment. The plastic film also induced the expression changes of TIA biosynthesis pathway genes. The further correlation analysis between alkaloid content and these genes transcriptional levels indicated that VIN accumulation was significantly correlated with the gene expression of desacetoxyvindoline-4-hydroxylase (D4h) and deacetylvindoline-4-O-acetyl transferase (Dat), and CAT accumulation was significantly correlated with strictosidine synthase (Str) gene expression (p<0.05). Therefore, the low light treatment with plastic film accelerated the accumulation of VBL in the leaves of C. roseus seedlings, which may have practical significance in the production of VBL.

Keywords: Catharanthus roseus; Gene expression; Low light; Plastic film; TIAs.

INTRODUCTION

Catharanthus roseus (L.) G. Don (Madagascar periwinkle), an important medicinal plant that produces more than 120 kinds of terpenoid indole alkaloids (TIAs) during secondary metabolites. The leaves of this species contain the alkaloids vinblastine (VBL) and vincristine (VCR), used in anticancer treatments. The VBL biosynthetic pathway is proposed by Kutney (1990), as a coupling product of vin-doline (VIN) and catharanthine (CAT). C. roseus produces only very small amounts of VBL, however, and despite concentrated efforts to increase this amount, only minimal improvements have been made.

Terpenoid indole alkaloids (TIAs) metabolites biosynthesis must accommodate the primary metabolic pathways

in plants; the primary metabolites are regulated by strict expression of biosynthetic pathways genes. The two early and two late TIAs biosynthetic pathway genes, tryptophan decarboxylase (Tdc), strictosidine synthase (Str), desace-toxyvindoline-4-hydroxylase (D4h) and deacetylvindoline 4-O-acetyl transferase (Dat) have been isolated, partially characterized, and over-expressed to improve the biosynthesis (El-Sayed and Verpoort, 2007), but the expression of these genes (Tdc, Str, D4h and Dat) in C. roseus seedlings under low light are little reported.

Regulation of TIAs metabolites biosynthesis can be influenced by developmental hormones and light. Tang et al. (2007) have reported the presence of low light-affected alkaloids in the leaves of C. roseus seedlings. Although the use of low UV light has increased the production of VIN and CAL in C. roseus tissue and cell cultures, this technique has not been effective in producing the important anticancer compound VBL (Ramani and Jayabaskara,

*Corresponding author: E-mail: arrive 100@163.com; Tel: +086-045182191517; Fax: +086-045182102082.

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2008). In our study, the quantities of VIN, CAT and VBL were measured in the leaves of aseptic C. roseus seedlings under plastic film as compared to fluorescent light, and the expression levels of 4 genes (Tdc, Str, Dat and D4h) related to TIAs biosynthetic pathway were examined by semi-quantitative RT-PCR (SQ-RT-PCR) under low light/UV light. Furthermore, we analyzed the pattern of correlation between gene expression and alkaloid accumulation and discussed the potential of four gene expressions in screening TIAs accumulation.

MATERIALS AND METHODS Plant material and treatment

Catharanthus roseus seeds were sterilized with 75% alcohol for 20 sec, sterilized with 6% NaClO for 20-30 sec: washed 3-5 times with aseptic water and soaked in 0.1% plant agar cultivation solution. Seeds were then cultured on Murashige Skoog (MS) medium with pH 5.8 at 25°C-28°C under 2000 lx irradiance (provided by fluorescent lamp, 40 W, Shanghai, China) with 16/8 h day/night pho-toperiod. The cultures were subcultivated weekly. Aseptic seedling on MS medium were maintained in 200 mL flake 1/2 MS medium (Chen and Li, 2009), supplemented with 2% (w/v) sucrose and 0.8% (w/v) agar without auxins, each flake containing 1 seedling. Seedlings at the 40-day-old stage were shaded by plastic film and kept in the shade for 30 days. The plastic film was replaced with new film twice weekly to avoid any degradation. Radiant energy at different regions of the spectrum was measured by a portable spectroradiometer (AvaSpec-2048-2-USB2, Amsterdam, Holland). The wavelengths under fluorescent light and plastic film were scanned at intervals of 1 nm, respectively. Leaves were collected and their alkaloid and chlorophyll contents measured at the same time of day on 0 d, 3 d, 7 d, 15 d and 30 d. For the SQ-RT-PCR experiment, the leaf samples were collected during the shading treatment at 0 h, 3 h, 6 h, 12 h, 24 h and 72 h, The gene expressions were determined between the control and treatment.

Alkaloid extraction and HPLC analysis

The analysis of alkaloids, including leaf content of VIN, CAT and VBL, was performed according to Luo et al. (2005). Fresh leaf powder (0.3 g) was dissolved in 10 mL absolute methanol (analytical grade). Low frequency ultrasonic technology (250 W, 40 kHz) was used to extract the alkaloids for 20 min. The methanol extract was centri-fuged at 21,000 g for 10 min and concentrated to 1 mL for high performance liquid chromatography (HPLC, Jasco, VG, Tokyo, Japan). The HPLC system was equipped with a Waters ODS C18 reversed-phase column (250x4.6 mm, 5 (im) and a photodiode-array detector set up at 220 nm. The sample injection volume was 10 (iL at a flow-rate of 1.5 mL min^-1.

Chlorophyll content analysis

The contents of chlorophyll a and b in the samples

under plastic film and fluorescent light treatment were determined according to Arnon (1949). Total chlorophyll, chlorophyll a and chlorophyll b contents were calculated from the absorbance at 665 and 649 nm.

RNA extraction and semi-quantitative RT-PCR (SQ-RT-PCR)

Total RNA was extracted from the leaves under plastic film using Trizol reagent (Shanghai Sangon, Shanghai China). Tdc, Str, D4h and Dat SQ-RT-PCR analysis were performed according to Marone et al. (2001) and (Oliveira and Lucas, 2004). cDNA was prepared from total RNA (2 ig) using RevertAid H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, Burlington, Canada), following manufacturer's instructions. Multiplex PCR reactions were performed with 2 (il of cDNA as a tem-plate, 50 mM MgCl2, 10 mM dNTP mix, 10 (imol of each primer, 4 (l of Actin primer, 2 units of Platinum Taq DNA polymerase (which enables a "hot-start" method to im-prove specificity of annealing), 20 mM Tris-HCl (pH 8.4) and 50 mM KCl in a total volume of 50 pi The primers used in this work were as follow:

-Tdc: 5'-ACACCACTAGCGAGTCCATT-3' and

5'- CCAACAGCCAGTCTTAGCAT-3' (GenBank Acc. No. X67662)

-Str: 5'- CCTTCCTATGCTCCGAATGC-3' and

5'- CCATCGTGCTCTTGAATCTG-3' (GenBank Acc. No. X61932)

-D4h: 5'- ACTATCAGAAGCTTTGGGGC-3' and

5'- GGGGTACCTCTAATGTTCACCG-3' (GenBank Acc. No. U71604)

-Dat: 5'- GAAGTTCGAATTCGTTGCCG-3' and

5'- GCTCTAGAAGTAGCTGTTCGTTC-3' (Gen-Bank Acc. No. AF053307)

-Actin: 5'- GGCTGGATTTGCTGGAGATGAT-3'

5'- TAGATCCTCCGATCCAGACACTG-3' (Gen-Bank Acc. No. AK322149)

Actin gene was used as an internal control. Amplification was conducted with 28-35 cycles of PCR reaction of 30 sec denaturation at 94°C, 30 sec annealing at 45-50°C, and 1 min extension at 72°C. PCR products were electrophore-sed on 1.5% agarose gel containing ethidium bromide, and were viewed under UV illumination. Pictures of the resultant PCR bands were digitized and quantified by den-sitometric analysis using Quantity One software (Bio-Rad, Hercules, CA, USA). Results presented were mean values of at least three independent experiments.

Data analysis

All data were subjected to one-way analysis of variance (ANOVA) and correlation analysis in the SPSS statistical package (IBM, Chicago, USA). The experiments were repeated three times and the mean and standard deviations were calculated.

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RESULTS

Spectra and radiation of the plastic film

The plastic film induced a low light/UV light environment compared to the fluorescent light (300 nm-1,100 nm). As shown in Table 1, UV radiation under plastic film only reached 6% of that under fluorescent light, and the total radiation under fluorescent light was higher (8.31 W m^-2) than that under plastic film (0.769 W m^-2).

Alkaloid content analysis under low light

The amount of VIN and CAT present in leaves initially showed a slight increase, reaching its peak (0.32 ±0.07 mg g^-1 FW and 0.63 ±0.18 mg g^-1 FW) on the 3rd day of the treatment. This was followed by a significant decrease (p<0.05) over 7 days of treatment, (Figure 1a and b). The change of dimeric alkaloid VBL content in treatment showed significant difference from that under fluorescent light (p<0.05), the VBL content became higher than that of control, with its peak value around 0.028 ±0.0051 mg g^-1FW on the 15th day (Figure 1c).

Chlorophyll content analysis under low light

The total chlorophyll content in control leaves gradually increased on the first 7 days and then decreased, whereas plastic film treatment resulted in a little increase of the chlorophyll content on the first 3rd day, and significant decrease (p<0.05) after 3 day of treatment, the lowest value (1.71±0. 35 mg g^-1 FW) was recorded on the 7th day (Figure 2a). The ratio of chlorophyll a and chlorophyll b in treatment leaves was consistently lower than that under fluorescent light (Figure 2b).

Gene expression analysis under low light

The mRNA levels of Tdc, Str, D4h and Dat were mea-

sured in leaves under fluorescent light and plastic film, As shown in Table 2, significant differences in gene expression were observed between the treatment and the control. There were significant increases in Tdc mRNA levels between 3 h and 6 h during shading treatment. Str expression was significantly up-regulated at 72 h during shading culture. Furthermore, the transcription levels of D4h and Dat were consistently higher in the treatment than in the control, and mRNA levels of D4h and Dat were signifi-

Figure 1. Effect of plastic film on the amount of vindoline (VIN), catharanthine (CAT) and vinblastine (VBL) in the leaves of aseptic C. roseus seedlings. Data shown are the mean and standard deviation (*p<0.05, **p<0.01; □, control;國，red film).

Table 1. Spectra and radiant energy of control and the plastic film (W m^-2).


Light Spectra	Ultraviolet 300-400 nm	Blue 400-510 nm	Green 510-610 nm	Red 610-720 nm	Near infrared 720-1,100 nm	Total radiation 300-1,100 nm
The plastic film	0.0173^b (6.0)	0.0483^b (1.66)	0.101^b (2.97)	0.432^b (39.6)	0.203^b (34.1)	0.769^b (9.3)
Control	0.288^a	2.914^a	3.405^a	1.106^a	0.596^a	8.31^a

Values in parenthesis are the percents of radiant energy of different light wave bands in overall radiant energy. Means within a column followed by the same letters are not significantly different by SPSS analysis p<0.05. W m^-2 is light energy irradiation per meter square.

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Table 2. Expression of Tdc, Str, D4h and Dat genes in leaves cultivated and collected between treatment and control at different hours (SQ-RT-PCR arbitrary units ± standard deviation). Means were compared to the 0 h value, which was set as 1.


Genes		Control (fluorescent light)		Treatment (plastic film)
Genes	0 h	3 h 6 h 12 h 24 h 72 h	3 h	6 h 12 h 24 h 72 h
Tdc	1.00±0.23	3.05±0.25 3.81±0.12 6.32±0.34 15.90±0.43 3.28±0.18	13.74±0.8914.20±0.68 4.56±0.56 13.32±1.23 5.90±0.45
Str	1.00±0.20	3.55±0.28 3.67±0.32 2.27±0.25 1.30±0.11 0.42±0.4	3.99±0.22	1.54±0.12 0.39±0.07 1.54±0.21 4.69±0.53
D4h	1.00±0.36	8.98±0.68 5.07±0.45 0.78±0.11 2.40±0.19 1.99±0.29	9.54±0.87	5.24±0.25 3.91±0.19 2.79±0.28 2.51±0.23
Dat	1.00±0.19	0.77±0.14 0.83±0.33 0.82±0.9 0.70±0.11 0.39±0.15	7.00±0.69	3.46±0.42 2.82±0.30 3.20±0.38 8.73±0.33

cantly up-regulated at 12 h and 72 h during shading culture (p<0.01). Thus, it is clear that low light/UV light activated the high expression of TIA pathway genes (Tdc, Str, D4h and Dat) at different hours.

Correlation analysis

Correlation analyses of alkaloid accumulation and gene expression (Table 3) show that there were significant (p<0.05) correlations between Str gene expression and CAT content, and between D4h and Dat gene expression and VIN content under plastic film treatment. No significant correlation was observed, however, between gene expression and the related alkaloid accumulation.

DISCUSSION

Light spectrum, intensity and quality play important roles in plants' development and metabolism processes. Dai et al. (2004) reported that plastic film could alter not only light intensity but also light spectrum and quality, and that low light induced by plastic film could increase camp-tothecin content in the leaves of Camptotheca acuminata seedlings. Qin et al. (2005) found that plastic film was conducive to strawberry growth and development in ex-plant culture. In addition, low UV lighting was reported to increase the levels of dimeric alkaloids while concurrently decreasing those of VIN and CAT (Hirata et al., 1993). In the study, lower UV radiation may accelerate plant growth and improve different alkaloids accumulation, because film's emission of different spectra lights (300 nm-1,100 nm) may overshadow the hazardous effects of UV radia-tion (Qin et al., 2005). On the other hand, plastic films as shade material could improve the temperature and humid-ity (Zhang and Xu, 2008), which may activate the VBL synthesis coupling VIN with CAT and increase VBL con-tent.

Light controls the development of plant chloroplasts and the biosynthesis of many important precursors for TIAs in developed chloroplasts (Zhao et al., 2001). The decrease of chlorophyll a/b ratio in the treatment is similar to what has been described for strawberry plants under plastic film (Qin et al., 2005). This may be due to an increase of chlorophyll degradation or to a decrease of chlorophyll synthesis, which affected the development of plant chloroplasts and the biosynthesis of important precursors,

Figure 2. Effect of plastic film on the total amount of chlorophyll and the chlorophyll a/b ratio in the leaves of aseptic C. roseus seedlings. Data shown are the mean and standard deviation (*p<0.05, **p<0.01; □, control; ■, red film).

Table 3. Correlation coefficient (r) between gene expression and alkaloid accumulation between control and treatment


Genes	Control (fluorescent light)	Treatment (plastic film)
Genes	VIN CAT	VIN CAT
Tdc	0.264	0.264
Str	0.461	0.934*
D4h	-0.157	0.933*
Dat	-0.084	0.903*

*Is significant at p<0.05 in ANOVA analysis.

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thus limiting the amount of substrate necessary for VBL biosynthesis. This may explain the initial increase in VBL content and its later decrease under shaded conditons (Figure 1c).

De Luca and Laflamme (2001) reported that alkaloid biosynthesis is regulated primarily at the level of gene expression. Tdc expression pattern is development-specific (Fernandez et al., 1989). In the experiment, Tdc expression was earlier up-regulated under the film. The effect of UV irradiation on expression of Tdc and Str, and CAT accumulation has been reported previously in C. roseus leaves (Ouwerkerk et al., 1999). Low UV irradiation has activated the transcription factors of the signaling pathway and improved Tdc and Str genes expression (Ramani and Chelliah, 2007). In the experiment, Str gene expression were up-regulated, and there were correlation of Str gene expression with CAT accumulation. On the other hand, light induction of D4h and Dat genes expression is mediated by phytochrome, and phytochrome is involved in the regulation of VIN biosynthesis (Aerts and De Luca, 1992). In the study, low light may change phytochrome, thus, D4h and Dat genes expression were activated and up-regulated, a positive correlation between D4h and Dat gene transcript abundance and VIN accumulation has been observed (Table 3). Therefore, we were able to provide a rapid method for estimating periwinkle alkaloids by analyzing their expression of TIAs biosynthetic pathway genes. The key to successful metabolite production in cultivated C. roseus is the establishment of an efficient culture system. Further studies need to be carried out that explore the mechanism of the culture system under the film.

Acknowledgements. The work was supported by the National Natural Science Foundation of China (No.30770338).

LITERATURE CITED

Aerts, R.J. and V. De Luca. 1992. Phytochrome is involved in the light regulation of vindoline biosynthesis in Catharan-thus. Plant Physiol. 100: 1029-1032.

Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:

1-10.

Chen, Q. and J.G. Li. 2009. Rapid propagation techniques of Catharanthus roseus (L.) G. Don. J. Anhui Agri. Sci. 37: 10404-10406.

Dai, S.J., Y. Wang, X.F. Yan, and M.F. Ma. 2004. Effects of color films on growth and camptothecin content in the leaves of Camptotheca acuminata seedlings. Acta Ecol. Sin. 24: 869- 875.

De Luca, V and P. Laflamme. 2001. The expanding universe of alkaloid biosynthesis. Curr. Opin. Plant Biol. 4: 225-233.

El-Sayed, M. and R. Verpoort. 2007. Catharanthus terpenoid indole alkaloids: biosynthesis and regulation. Phytochem. Rev. 6: 277-305.

Fernandez, J.A., T.G. Owen, W.G.W. Kurz, and V. De Luca. 1989. Immunological detection and quantification of tryp-tophan decarboxylase in developing Catharanthus roseus seedlings. Plant Physiol. 91: 79-84.

Hirata, K., M. Asada, E. Yatani, K. Miyamoto, and Y. Miura. 1993. Effect of near ultraviolet light on alkaloid production in Catharanthus roseus plants. Planta Med. 59: 46-50.

Kutney, J.P. 1990. Biosynthesis and synthesis of indole and bisindole alkaloids in plant cell cultures: a personal overview. Nat. Prod. Rep. 7: 85-103.

Luo, M., Y.J. Fu, Y.G. Zu, Q. Shi, F.S. Mu, and Q.Y. Li. 2005.

Rapid determination of four vinca alkaloids by reversed phase high performance liquid chromatography. Chin. J.

Anal. Chem. 33: 89-92.

Marone, M., S. Mozzetti, D. De Ritis, L. Pierelli, and G. Scam-bia. 2001. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biol. Proced. 3: 19-25.

Oliveira, R. and C. Lucas. 2004. Expression studies of GUP1 and GUP2, genes involved in glycerol active transport in

Saccharomyces cerevisiae, using semi-quantitative RT-

PCR. Curr. Genet. 46: 140-146.

Ouwerkerk, P.B.F., D. Hallard, R. Verpoorte, and J. Memelink. 1999. Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus. Plant Mol. Biol. 41: 491-503.

Qin, Y.H., S.L. Zhang, A. Syed, L.X. Zhang, Q.P. Qin, K.S. Chen, and C.J. Xu. 2005. Regeneration mechanism of Toyo-noka strawberry under different color plastic films. Plant Sci. 168: 1425-1431.

Ramani, S. and C. Jayabaskaran. 2008. Enhanced catharanthine and vindoline production in suspension cultures of Cathar-anthus roseus by ultraviolet-B light. J. Mol. Signal. 3: 9-15.

Ramani, S. and J. Chelliah. 2007. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures. B.M.C. Plant

Biol. 7: 61-78.

Tang, Z.H., X.R. Guo, J.H. Yu, L. Yang, Y.F. Sun, and Y.G. Zu.

2007. Effects of low light intensity on changes of soluble sugars, alkaloids and phytohormones in Catharanthus

roseus seedlings. Acta Ecol. Sin. 27: 4419-4424.

Zhang, R.H. and K. Xu. 2008. Effects of shading light quality at seedling stage on the photosynthesis and growth of ginger.

Chin. J. App1. Eco1. 19: 499-504. Zhao, J., W.H. Zhu, and Q. Hu. 2001. Effects of light and plant

growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures.

Plant Growth Regul. 33: 43-49.

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低光輻射對長春花葉片中吲哚生物鹼含量積累及相關基因表達的影響

劉英^1,2 趙冬梅^1,2 祖元剛^1,2 唐忠華^1,2 張衷華^1,2 姜揚^1,2 史典義³

¹中國哈爾濱東北林業大學森林植物生態學教育部重點實驗室
²中國哈爾濱東北林業大學林業生物製劑教育部工程研究中心
³中國北京中國農業大學玉米改良研究中心

長春花(Catharanthus roseus)作為重要的藥用植物，以其生產的次生代謝物質長春鹼
(Vinblastine, VBL)和長春新鹼(Vincristine, VCR)具有重要的抗癌作用而聞名。在塑料膜誘導的一個
低光/低紫外光環境下，研究長春花幼苗葉片中葉綠素含量，生物鹼含量以及萜類吲哚生物鹼(Terpe-
noid indole alkaloids, TIAs)合成路徑相關基因表達的變化，結果表明：葉綠素a/b比率一直低於日光
燈下葉綠素的a/b比率。與對照相比，葉綠素含量先升高然後明顯下降（p<0.05)。同時，處理的葉片
中文多靈（Vindoline, VIN)和長春質鹼（Catharanthine, CAT)的含量稍有升高後低於對照，但是，耦
合VIN和CAT的產物VBL的含量逐漸升高，在處理第15天達到最高（0.028 mg g^-1 FW±0.0051)。
此外，塑料遮光膜誘導了 VBL的合成路徑相關基因的表達變化，相關性分析表明：VIN的積累與脫
乙酰文多靈-4-羥化酶（D4/2)和脫乙酰文多靈-4-0-乙酰轉移酶(DAT)的基因表達均呈顯著相關性
（p<0.05)，而CAT的積累與異胡豆苷合成酶(Str)的基因表達呈顯著相關。因此，本研究採用濾光膜遮
光處理促進VBL在幼苗葉片中的積累，對VBL的生產具有重要實踐意義。

關鍵詞：萜類吲哚生物鹼；長春花；基因表達；低光；塑膠膜。



	196	Botanical Studies, Vol. 52, 2011

	低光輻射對長春花葉片中吲哚生物鹼含量積累及相關基因表達的影響劉英^1,2 趙冬梅^1,2 祖元剛^1,2 唐忠華^1,2 張衷華^1,2 姜揚^1,2 史典義³ ¹中國哈爾濱東北林業大學森林植物生態學教育部重點實驗室 ²中國哈爾濱東北林業大學林業生物製劑教育部工程研究中心 ³中國北京中國農業大學玉米改良研究中心長春花(Catharanthus roseus)作為重要的藥用植物，以其生產的次生代謝物質長春鹼 (Vinblastine, VBL)和長春新鹼(Vincristine, VCR)具有重要的抗癌作用而聞名。在塑料膜誘導的一個低光/低紫外光環境下，研究長春花幼苗葉片中葉綠素含量，生物鹼含量以及萜類吲哚生物鹼(Terpe- noid indole alkaloids, TIAs)合成路徑相關基因表達的變化，結果表明：葉綠素a/b比率一直低於日光燈下葉綠素的a/b比率。與對照相比，葉綠素含量先升高然後明顯下降（p<0.05)。同時，處理的葉片中文多靈（Vindoline, VIN)和長春質鹼（Catharanthine, CAT)的含量稍有升高後低於對照，但是，耦合VIN和CAT的產物VBL的含量逐漸升高，在處理第15天達到最高（0.028 mg g^-1 FW±0.0051)。此外，塑料遮光膜誘導了 VBL的合成路徑相關基因的表達變化，相關性分析表明：VIN的積累與脫乙酰文多靈-4-羥化酶（D4/2)和脫乙酰文多靈-4-0-乙酰轉移酶(DAT)的基因表達均呈顯著相關性（p<0.05)，而CAT的積累與異胡豆苷合成酶(Str)的基因表達呈顯著相關。因此，本研究採用濾光膜遮光處理促進VBL在幼苗葉片中的積累，對VBL的生產具有重要實踐意義。關鍵詞：萜類吲哚生物鹼；長春花；基因表達；低光；塑膠膜。