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692 生物工程学报 Chinese Journal of Biotechnology http://journals.im.ac.cn/cjbc

1 692 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 李翠萍等 / 转番茄 GGPS2 基因烟草的构建及弱光耐受性分析 May 25, 2015, 31(5): Chin J Biotech, All rights reserved 农业生物技术 GGPS2 李翠萍 1, 董卫华 1, 张兴国 ,,. GGPS2., 2015, 31(5): Li CP, Dong WH, Zhang XG. Construction of transgenic tobacco expressing tomato GGPS2 gene and analysis of its low light tolerance. Chin J Biotech, 2015, 31(5): 摘要 : 为探讨弱光处理对转番茄 Solanum lycopersicon L. GGPS2 基因烟草的类胡萝卜素叶绿素合成及耐弱光性的影响, 将 SlaGGPS2 基因和绿色荧光蛋白报告基因 (GFP) 经农杆菌介导转入烟草 Nicotiana tabacum L. cv. Wisconsin 38 PCR 检测证明抗卡那霉素烟草含有 nptii SlaGGPS2 基因, 且无农杆菌污染 ; 荧光检测发现, 抗卡那霉素烟草的根尖呈现特有的荧光, 由此说明获得了整合 SlaGGPS2 和 GFP 等外源基因的转基因烟草弱光处理后, 发现转 SlaGGPS2 基因烟草的类胡萝卜素含量叶绿素总量光合速率单位叶面积重总干重根冠干重比均比野生烟草高, 达到了差异显著水平证实 SlaGGPS2 基因增加了弱光下烟草的类胡萝卜素含量叶绿素总量, 增强了光合速率, 促进了生物量积累及其向根部的分配, 提高了烟草弱光下的耐受性, 推测可用于其他作物的耐弱光性改良 : 烟草, 番茄 GGPS2 基因, 类胡萝卜素, 叶绿素, 光合速率, 弱光耐受性 Received: October 1, 2014; Accepted: December 15, 2014 Supported by: the Doctoral Fund of Xinxiang Medical College (No ). Corresponding author: Cuiping Li. Tel: ; lcp831220@163.com (No )

2 李翠萍等 / 转番茄 GGPS2 基因烟草的构建及弱光耐受性分析 693 Construction of transgenic tobacco expressing tomato GGPS2 gene and analysis of its low light tolerance Cuiping Li 1, Weihua Dong 1, and Xingguo Zhang 2 1 Department of Biochemistry and Molecular Biology, Xinxiang Medical College, Xinxiang , Henan, China 2 Key Laboratory of Horticulture Science for Southern Mountainous Regions, College of Horticulture and Landscape Architecture, Southwest University, Chongqing , China Abstract: To explore the influence of low light on the synthesis of carotenoids, chlorophyll and the adaptability of transgenic plants with tomato Solanum lycopersicon L. GGPS2 gene, we constructed a vector containing a GGPS2 gene with green fluorescent protein (GFP) as report gene under the control of a cauliflower mosaic virus 35S promoter and introduced it into tobacco Nicotiana tabacum L. cv. Wisconsin 38 by Agrobacterium tumefaciens-mediated transformation. PCR analysis of the DNA from kanamycin resistant tobacco indicated that the transgenic tobacco containing the nptii gene, SlaGGPS2 gene and without contamination of Agrobacterium. We also detected the root tip of kanamycin resistant tobacco showing characteristic fluorescence. The contents of carotenoid, chlorophyll and photosynthesis of transgenic tobacco increased in comparison with wild tobacco after low light treatment. In addition, leaf mass per unit area, total dry weight, ratio of root to shoot in transgenic tobacco were all higher than that of the wild tobacco, which proved that the transgenic tobacco could increase the accumulation of biomass and promote it transport to root. The transgenic tobacco with SlaGGPS2 gene can increase the contents of carotenoid, chlorophyll, enhance the photosynthetic rate, promote the biomass accumulation and its distribution to root. Hence, the transgenic tobacco with SlaGGPS2 gene had increased low light tolerance and the SlaGGPS2 gene maybe can be used in other crops. Keywords: tobacco, the GGPS2 gene from tomato, carotenoids, chlorophyll, photosynthesis, low light resistant [1-3] 牻牻 (GGPP) E [4-7] [8-9] 牻牻 (Geranylgeranyl pyrophosphate synthase GGPS) [10-11] GGPS [10] [12] 5 GGPS AtGGPS1 AtGGPS3 AtGGPS2 AtGGPS4 AtGGPS6 GGPP [12] 2 GGPS GGPS1 mrna GGPS2 [13] LeGGPS2 GGPP (PSY) (crti) [14] IPI GGPS β- [15] GGPS cjb@im.ac.cn

3 694 ISSN CN /Q Chin J Biotech May 25, 2015 Vol.31 No.5 GGPS [16-17] GGPS CaMV 35S GGPS2 1 材料与方法 1.1 材料 Solanum lycopersicon L. cv Ailsa Craig Nicotiana tabacum L. cv. Wisconsin 38 Escherichia coli XL1-blue Agrobacterium tumefaciens EHA105 Plant RNAzol pet32a(+) Novagen pvct2024 SanPrep DNA PrimStar HS DNA Taq DNA DNA marker TaKaRa 1.2 植物表达载体的构建 SlaGGPS2 cdna [18] pvct 烟草的遗传转化 35S-SlaGGPS2-Tnos EHA105 [19] MS 2.5 mg/l 6-BA 0.2 mg/l NAA 150 mg/l 300 mg/l 3% 0.65% MS 150 mg/l 300 mg/l 3% 0.65% 1.4 转基因植株的鉴定 PCR CTAB DNA gdna 20 pvct2295 DNA P1 P2 P1 P3 P4 P5 PCR SlaGGPS GFP 1 cm (D5000B Leica ) 470 nm 表 1 引物序列 Table 1 Primer sequences Primer name Sequences (5 3 ) P1 GAATTCAGGGAGTCACGTTATGACC P2 CCCGGCAGCTTAGTTGCCGT P3 CATGAGCGAAACCCTATAGGAACC P4 CGGGATCCATATGAGATCTATGAACCTT GT P5 GCTAGCTTAATTTTGACGATTAGCAATG

4 李翠萍等 / 转番茄 GGPS2 基因烟草的构建及弱光耐受性分析转基因烟草的耐弱光性检测 μmol/(m 2 s) 3 (TG1 TG2 TG3) (WT) 3 3 T5 14 h /10 h μmol/(m 2 s) 50 d μmol/(m 2 s) 30 d 60 d a b μmol/(m 2 s) 50 d μmol/(m 2 s) 30 d 5 9:00 11:00 LI-6400 (LI-COR ) 60 μmol/(m 2 s) mm 5 ( 8 11 ) ml 80% r/min [20] UV nm 646 nm 663 nm a (C a ) b (C b ) (C T ) (C c ) C a =(12.21 A A 646 ) C b =(20.13 A A 663 ) C T = C a + C b C c =(1 000 A C a 104 C b )/229 C V/S A C V S (YC/T ) [21] min μmol/(m 2 s) 50 d μmol/(m 2 s) 90 d Excel SPSS 结果与分析 2.1 得到整合有 SlaGGPS2 等基因的烟草植株 SlaGGPS2 ( 1A) 33 Kan ( 1B E) PCR 9 Kan ( 1F G) DNA ( 1H) Kan GFP ( 1I L) nptii SlaGGPS2 GFP 9 cjb@im.ac.cn

696 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech May 25, 2015 Vol.31 No.5 图 1 SlaGGPS2 基因转烟草植株的鉴定 Fig. 1 Identification of transgenic tobacco with SlaGGPS2 gene.

5 696 ISSN CN /Q Chin J Biotech May 25, 2015 Vol.31 No.5 图 1 SlaGGPS2 基因转烟草植株的鉴定 Fig. 1 Identification of transgenic tobacco with SlaGGPS2 gene. (A) The structure of T-DNA in binary vector containing SlaGGPS2 gene. (B) The infected tobacco leaves. (C) antibiotic buds. (D) Rooted resistant tobacco. (E) Tobacco after transplanting. (F H) The PCR products of nptii gene, SlaGGPS2 gene, the left border of T-DNA. (I J) The root tip of wild tobacco in white and blue light respectively. (K L) The root tip of transgenic tobacco in white and blue light respectively. Ms: DL2000 DNA marker; Mb: λdna/ecorⅠ+ Hind Ⅲ marker; 1 9: the transgenic plants; : non-transgenic plants as negative control; +: binary vector as positive control; bar=100 μm. 转 SlaGGPS2 基因能增强弱光下烟草的 2.2 光合性能和生物量积累不同弱光处理对转 SlaGGPS2 基因烟草光合性能的影响合速率较野生烟草增加 25% 35.5% 均达到差异显著水平 (P<0.05) (表 2) μmol/(m2 s) 弱光照处理 30 d 后转基因烟草株系 TG1 和 TG2 第 5 功能叶的类胡萝 μmol/(m s) 弱光照处理 50 d 后转卜素含量分别比野生烟草增加 18% 16% 叶绿基因烟草株系 TG1 和 TG2 第 5 功能叶的类胡萝素总量分别比野生烟草增加 23% 25% 达到差卜素含量分别比野生烟草增加 13% 4% 叶绿异显著水平 (P<0.05) 而株系 TG3 的类胡萝卜素总量分别比野生烟草增加 19% 12% 而株系素含量比野生烟草低 3% 叶绿素总量比野生烟 TG3 的类胡萝卜素含量与野生烟草相当叶绿草低 4% 所有转基因烟草株系第 5 功能叶的光素总量比野生烟草增加 12% 均未达到差异显合速率较野生烟草增加 1.8% 11.7% 未达到差著水平所有转基因烟草株系第 5 功能叶的光异显著水平 (表 2)

6 李翠萍等 / 转番茄 GGPS2 基因烟草的构建及弱光耐受性分析 SlaGGPS μmol/(m 2 s) 60 d TG1 TG2 5 6% 5% 5% 3% TG3 4% 8% 5 2% 8% ( 3) 表 2 不同弱光处理对转 SlaGGPS2 基因烟草色素含量和光合速率的影响 Table 2 Effects of different low light treatments on pigment contents and photosynthetic rate of transgenic tobacco with SlaGGPS2 gene Lines Light intensity (μmol/(m 2 s)) Processing time (d) Carotenoid content (μg/cm 2 ) Chlorophyll content (μg/cm 2 ) Photosynthetic rate (μmol CO 2 /(m 2 s)) WT ± ± ±0.7 TG1 4.9± ± ±1.2 * TG2 4.6± ± ±0.4 * TG3 4.4± ± ±1.2 * WT ± ± ±0.6 TG1 5.1±0.4 * 31.1±4.2 * 1.9±0.5 TG2 5.0±0.8 * 31.5±6.6 * 1.9±0.4 TG3 4.2± ± ±0.6 WT: wild tobacco; TG1, TG2, TG3: different lines of transgenic tobacco; values are x ±s (n=3); ** and * indicate 1%, 5% significant level respectively. 表 3 弱光处理对转 SlaGGPS2 基因烟草不同部位叶片色素含量和光合速率的影响 Table 3 Effects of low light treatment on pigment contents and photosynthetic rate of different location of leaves in transgenic tobacco with SlaGGPS2 gene Lines Different location of leaves (from top to bottom) Carotenoid content (μg/cm 2 ) Chlorophyll content (μg/cm 2 ) Photosynthetic rate (μmol CO 2 /(m 2 s)) WT 5 5.5± ± ±0.6 TG1 5.8± ± ±0.4 TG2 5.8± ± ±0.6 TG3 5.3± ± ±0.7 WT 8 5.2± ±3.0 ND TG1 6.2± ±2.3 ND TG2 5.4± ±2.3 ND TG3 4.7± ±3.5 ND WT ± ±3.4 ND TG1 4.8± ±2.1 ND TG2 4.6± ±2.1 ND TG3 4.4± ±3.2 ND WT: wild tobacco; TG1, TG2, TG3: different lines of transgenic tobacco; ND: not detected; values are x ±s (n=3). cjb@im.ac.cn

698 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech May 25, 2015 Vol.31 No.5 TG1 TG2 8 18% 4% 14% 9% TG3 10% 13% ( 3) 11 13% 23% 8% 21% ( 3) 2.2.3 SlaGGPS2 25 30 μmol/(m 2 s) 50 d 50 60 μmol/(m 2 s) 90 d TG1 TG2 12% 6% ( 2A) 14% 3% ( 2C) TG3 7% 5% ( 2A 2C) 6% 11% ( 2B) 14% 26% (P<0.

7 698 ISSN CN /Q Chin J Biotech May 25, 2015 Vol.31 No.5 TG1 TG2 8 18% 4% 14% 9% TG3 10% 13% ( 3) 11 13% 23% 8% 21% ( 3) SlaGGPS μmol/(m 2 s) 50 d μmol/(m 2 s) 90 d TG1 TG2 12% 6% ( 2A) 14% 3% ( 2C) TG3 7% 5% ( 2A 2C) 6% 11% ( 2B) 14% 26% (P<0.05) 5% 20% TG3 (P<0.05) TG3 2% 16% 55% 95% (P<0.01) TG1 (P<0.05) TG1 TG2 8% 5% TG3 1% 16% 47% 39% 67% 9% 26% (P<0.05) 图 2 弱光对转基因烟草叶片数目 (A) 茎粗 (B) 株高 (C) 的影响 Fig. 2 The leaf number (A), stem diameter (B), height (C) of transgenic tobacco under low light. 17% 28% (P<0.01) 28% 37% (P<0.01) ( 4)

8 李翠萍等 / 转番茄 GGPS2 基因烟草的构建及弱光耐受性分析 699 表 4 弱光下转 SlaGGPS2 基因烟草的生物量积累 Table 4 The biomass of transgenic tobacco with SlaGGPS2 gene under low light Lines LMA FW of FW of FW of DW of DW of DW of DW/FW Ration of root to (mg/cm 2 FW (g) DW (g) ) leave (g) stem (g) root (g) leave (g) stem (g) root (g) (%) shoot DW (%) WT 13.4± ± 36.3± 8.6± 134.9± 7.6± 3.2± 1.8± 12.6± 9.3± 17.0± TG1 16.9±3.8 * 85.1± 42.1± 16.8± 147.0± 8.2± 4.7± 3.0± 15.9± 10.8± 23.3±1.7 ** ** 3.6 * ** 0.2 ** 0.2 ** 0.3 ** TG2 16.7±2.8 * 83.4± 37.0± 14.2± 134.6± 8.0± 4.4± 2.7± 15.1± 11.2± 21.8±0.4 * ** ** 0.1 ** 0.4 ** 0.1 ** TG3 15.3±1.6 * 72.2± 2.5 * 28.8± 0.7 * 13.3± 0.2 ** 114.3± 3.2 ** 7.5± ± 0.1 * 2.5± 0.1 ** 13.7± 0.3 * 11.9± 0.1 ** 22.3±0.6 * WT: wild tobacco; TG1, TG2, TG3: different lines of transgenic tobacco; LMA: leaf mass per unit area; FW: fresh weight; DW: dry weight; values are x ±s (n=3); ** and * indicate 1%, 5% significant level respectively. 3 讨论 [23-24] GGPS GGPP 35S-SlaGGPS2-Tnos ( 1A) TG1 TG μmol/(m 2 s) 50 d 25% 35.5% (P<0.01) ( 2) μmol/(m 2 s) 30 d TG1 TG2 5 18% 16% 23% 25% (P<0.05) ( 3) GGPS TG3 Ji [16] GGPS 20% PSY ZDS Lintig [22] ( 3) GGPP TG1 TG μmol/(m 2 s) 50 d μmol/(m 2 s) cjb@im.ac.cn

9 700 ISSN CN /Q Chin J Biotech May 25, 2015 Vol.31 No.5 30 d ( 2 3) SlaGGPS [25] T1 PCR 3 1 SlaGGPS SlaGGPS REFERENCES [1] Barkey KO, Wells R. Response of soybean photosynthesis and chloroplast membrane function to canopy development and mutual shading. Plant Physiol, 1991, 97(1): [2] Chen QJ, Zhang FM, Wang YJ, et al. Studies of physiologic characteristics of reaction of cucumber to low temperature and poor light. Sci Agric Sin, 2003, 36(1): (in Chinese).,,,.., 2003, 36(1): [3] Ai XZ, Guo YK, Ma XZ, et al. Photosynthetic characteristics and ultrastructure of chloroplast of cucumber under low light intensity in solar green house. Sci Agric Sin, 2004, 37(2): (in Chinese).,,,.., 2004, 37(2): [4] Bartley GE, Scolnik PA. Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell, 1995, 7(7): [5] Koornneef M, Léon-Kloosterziel KM, Schwartz SH, et al. The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopsis. Plant Physiol Biochem, 1998, 36(1): [6] Andrew LP. Gibberellins in Arabidopsis. Plant Physiol Bioch, 1998, 36: [7] Wettstein DV, Gough S, Kannangara CG. Chlorophyll biosynthesis. Plant Cell, 1995, 7(7): [8] Bramley PM. Regulation of carotenoid formation during tomato fruit ripening and development. J Exp Bot, 2002, 531(377): [9] Oster U, Rudiger W. The G4 gene of Arabidopsis thaliana encode a chlorophyll synthase of etiolated plants. Bot Acta, 1997, 110(5): [10] Kuntz M, Romer S, Suire C. Identification of a cdna for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening. Plant J, 1992, 2(1): [11] Wu ZM, Zhang X, Wan JM. Molecular regulation of chlorophyll biosynthesis. Plant Physiol Comm, 2008, 44(6): (in Chinese).,,.., 2008, 44(6): [12] Okada K, Saito T, Nakagawa T, et al. Five

10 李翠萍等 / 转番茄 GGPS2 基因烟草的构建及弱光耐受性分析 701 geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis. Plant Physiol, 2000, 122(4): [13] Ament K, Van Schie CC, Bouwmeester HJ, et al. Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways. Planta, 2006, 224(5): [14] Chen JY, Pu ZQ, Xiao YW, et al. Lycopene synthesis via tri-cistronic expression of LeGGPS2, LePSY1 and CrtI in Escherichia coli. Chin J Biotech, 2012, 28(8): (in Chinese).,,,. LeGGPS2 LePSY1 CrtI., 2012, 28(7): [15] Sun J, Sun XX, Tang PW, et al. Molecular cloning and functional expression of two key carotene synthetic genes derived from Blakeslea trispora into E. coli for increased β-carotene production. Biotechnol Lett, 2012, 34(11): [16] Ji J, Wang G, Wang JH. Functional analysis of multiple carotenogenic genes from Lycium barbarum and Gentiana lutea L. for their effects on β-carotene production in transgenic tobacco. Biotechnol Lett, 2009, 31(2): [17] Kim M, Kim SC, Song KJ, et al. Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit. Plant Cell Rep, 2010, 29(12): [18] Li CP, Xia BB, Zhai JP, et al. Cloning and expression vector construction of the GGPS2 gene from tomato. J Southwest Univ: Nat Sci Ed, 2010, 32(10): (in Chinese).,,,. GGPS2. :, 2010, 32(10): [19] Horsch RB, Fry JE, Hoffman NL, et al. A simple and general method of transferring genes into plants. Science, 1985, 227(4691): [20] Xu DQ. Several problems in measurement and application of chlorophyll content. Plant Physiol Comm, 2009, 45(9): (in Chinese)..., 2009, 45(9): [21] State tobacco monopoly administration. Investigation Methods of Agronomical Character of Tobacco (YC/T 142). Beijing: China Standard Press, 1998 (in Chinese).. (YC/T 142). :, [22] Lintig JV, Welsch R, Bonk M, et al. Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J, 1997, 12(3): [23] Pattanayak GK, Biswal AK, Reddy VS, et al. Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants. Biochem Biophys Res Commun, 2005, 326(2): [24] Tanaka R, Koshino Y, Sawa S, et al. Overexpression of chlorophyllide a oxygenase enlarges the antenna size of photosystem II in Arabidopsis thaliana. Plant J, 2001, 26(4): [25] Li CP, Pan Y, Chu FT, et al. Construction of co-expression vector containing AtCAO and AtHEMA1 genes from Arabidopsis and transformation into tobacco. China Biotechnol, 2013, 33(4): (in Chinese).,,,. AtCAO AtHEMA1., 2013, 33(4): ( 本文责编郝丽芳 ) cjb@im.ac.cn

13期

13期中国农学通报,9():- Chinese Agricultural Science Bulletin 药百合种子萌发及染色体倍性检测吴昀, 马怡迪, 张琳, 夏宜平 ( 浙江大学农业与生物技术学院园艺系 / 农业部园艺植物生长发育与品质调控重点开放实验室, 杭州 ) 摘要