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1 44 1 Vol.44, No OCEANOLOGIA ET LIMNOLOGIA SINICA Jan., 2013 (Agrobacterium tumefaciens) (Chlorella vulgaris) * ( ) pcambia2301-idi, LBA4404, pcambia2301-idi T-DNA, G418 (NPT ), PCR idi NPT,, 0.84mg/g, 30.95%, 1.98mg/L, 36.77%,,, idi, Q943.2,, (, 2005) (Dwyer et al, 2001) (Carpentier et al, 2009),, (, 2010),,, (Fernandez- Sevilla et al, 2010),,, (Wang et al, 2007; Chen et al, 2001; Chow et al, 1999) (Dawson et al, 1997) PEG-CaCl 2 (Kim et al, 2002) (Cha et al, 2012),, DNA, Cha (2012) Chlorella vulgaris,, (Kumar et al, 2004) (Kathiresan et al, 2009) (Anila et al, 2011) (Cheng et al, 2012) (Cha et al, 2011) (IPP) (DMAPP) (, 2009) IPP IPP DMAPP, IPP (idi), (Chlorella vul- *, , maruijuan87@126.com :,,, xzlin@tiosoa.org : , :

2 garis) (Agrobacterium tumefaciens)lba4404 (Escherichia coli)dh5α pcambia (G418 ) (AS), ( ),, (FC ): 10g/L, 2g/L, 1g/L, 1g/L, FeSO 4 7H 2 O 2mg/L; LB : 10g/L, 5g/L, NaCl 10g/L; TYNG : 10g/L, 5g/L, NaCl 5g/L, MgSO 4 7H 2 O 0.2g/L; IM (Bundock et al, 1995; Hooykaas et al, 1979) DH5 idi, T (Takara) pbluescript II SK CAMV35S, CYC1 idi Hind BamH, NPT pcambia2301, pcambia2301-idi LBA OD , (Shen et al, 1989) idi pcambia2301-idi LBA4404, 50μg/ml 50μg/ml 100μg/ml TYNG 5ml TYNG ( 50μg/ml 50μg/ml 100μg/ml ), r/min 24h, 5ml 50ml TYNG OD , IM 1h, 4 5ml IM 10ml IM ( 0.2mol/L KCl 150μmol/L AS 50μg/ml ) 500μl, 4 5h 50ml FC, r/min,, 20ml (50mmol/L EDTA, 25mmol/L DTT, ph mmol/L ), 28 30min 0.2mol/L KCl, 10ml (2%, 2%, 0.2mol/L KCl), 28 14h, 0.2mol/L KCl IM, 2ml 0.2mol/L KCl IM 200μl, 10ml IM, 25 24h 500μl 0.2mol/L KCl IM, 450μg/ml G μg/ml 300μg/ml 0.2mol/L KCl FC, 28 FC,, PCR idi-f: 5 -ATGCAAACGGAACACGTC-3 ; idi-r: 5 -TTATTT AAGCTGGGTAAATGC-3 ; NPT II-F: 5 -TCACTGAA GCGGGAAGGGACT-3 ; NPT II-R: 5 -GCGGCGAT ACCGTAAAGCAC-3 7,, / DNA, idi-f/idi-r NPT II-F/NPT II-R PCR PCR : 94 5min, 94 45s, 58 1min, 72 1min 30s, 32, 72 15min % 100ml FC 250ml, r/min 5d 8000r/min 10min,,, g, 2.5ml 30min( 70%, 35 ), 1.5ml 20% KOH 10min 20ml, 5000r/min 5min,, 40, 5ml 5, 0.22μm HPLC DH5α 549bp idi, NCBI pbluescript II SK CAMV35S, CYC1 idi

3 1期马瑞娟等: 根癌农杆菌(Agrobacterium tumefaciens)介导培育富含叶黄素的基因工程小球藻(chlorella vulgaris)的研究 161 pcambia2301 质粒的多克隆位点, 构建了双元表达转化子的基因组 DNA 均能扩增出 idi 基因和 NPT II 载体 pcambia2301-idi(图 1) 经 PCR 扩增和酶切得基因, 而野生型小球藻的基因组 DNA 不能扩增出来到预期长度(1420bp)的 idi 基因表达模块片段, 证明 (图 3a, b), 说明目的基因已经插入小球藻的基因组中含有 idi 基因的载体 pcambia2301-idi 构建成功 2.4 小球藻生物量的测定图 4 所示为相同培养条件下野生型和转化子生农杆菌介导的转化小球藻与根癌农杆菌共培养后涂布于含 450μg/ml 物量由图 4 可以看出小球藻转化子 I1 I2 I3 I4 G μg/ml 氨苄青霉素 300μg/ml 头孢霉素和和 I5 的生物量与野生型无明显差异, 但 I6 和 I7 的生 0.2mol/L KCl 的 FC 培养基中, 28 培养 5 7d 后平板物量和野生型相比有所降低说明农杆菌介导 idi 基上有藻落长出(图 2a), 而未加农杆菌的对照组无藻落因的转化对大部分小球藻的生长没有显著影响长出(图 2b) 小球藻叶黄素的测定图 5 所示为每克小球藻干藻粉所含叶黄素的量小球藻转化子的鉴定转化子于不含抗生素的液体 FC 培养基中培养, 从图 5 中可以看出小球藻转化子 I1 I2 I3 和 I4 的长出后在筛选培养基上划线, 均能正常生长, 而野生叶黄素含量与野生型的差异较大, 其中, I4 的叶黄素 2.3 型小球藻不能生长(图 2c), 说明转化子是阳性转化子含量最高, 达到 0.84mg/g, 与野生型相比提高了酚/氯仿法提取转化子和野生型小球藻的基因组 30.95% 而 I5 I6 和 I7 的叶黄素含量则与野生型无 DNA, 以 idi-f/idi-r 和 NPT II-F/NPT II-R 引物扩增, 明显差异进一步分析每升藻液中叶黄素的产量, 发图1 Fig.1 双元载体 pcambia2301-idi 的构建 Construction of binary vector pcambia2301-idi

162 海洋与湖 44 卷沼现 I1 I3 和 I4 的叶黄素产量相对于野生型有一定提高, 其中 I4 的叶黄素产量最高, 达到 1.98mg/L, 比野生型提高了 36.

2 Screening positive clone of A. tumefaciens-mediated transformation of C. vulgaris a.

转化子于筛选平板进一步鉴定 3 讨论目前, 叶黄素的生物合成途径已经基本阐明, 一些关键酶基因已经分离出来, 利用基因工程技术提高微藻的叶黄素含量也取得了一定进展 Cordero

2 倍 Huang 等(2006)克隆了小球藻 Chlorella zofingiensis 的八氢番茄红素脱氢酶基因(PDS), 并使其成功在大肠杆菌中表达但是利用

3 PCR amplification to identify transformants a. PCR 扩增 idi 基因; b. PCR 扩增 NPT II 基因图5 Fig.

4 162 海洋与湖 44 卷沼现 I1 I3 和 I4 的叶黄素产量相对于野生型有一定提高, 其中 I4 的叶黄素产量最高, 达到 1.98mg/L, 比野生型提高了 36.77% I2 I5 和 I7 的叶黄素产量和野生型无明显差异, 而 I6 的叶黄素产量相对于野生型则有所降低(图 6) 图2 根癌农杆菌介导小球藻转化阳性克隆筛选 Fig.2 Screening positive clone of A. tumefaciens-mediated transformation of C. vulgaris a. 实验组: 小球藻与农杆菌共培养, 有藻落长出; b. 对照组: 小球藻未与农杆菌共培养, 无藻落长出; c. 转化子于筛选平板进一步鉴定 3 讨论目前, 叶黄素的生物合成途径已经基本阐明, 一些关键酶基因已经分离出来, 利用基因工程技术提高微藻的叶黄素含量也取得了一定进展 Cordero 等(2011)将小球藻的八氢番茄红素合成酶基因(PSY)克隆出来并转入莱茵衣藻中, 使衣藻细胞中八氢番茄红素合成酶过量表达, 结果发现转化子叶黄素的含量提高了 2.2 倍 Huang 等(2006)克隆了小球藻 Chlorella zofingiensis 的八氢番茄红素脱氢酶基因(PDS), 并使其成功在大肠杆菌中表达但是利用基因工程技术改造小球藻以提高叶黄素含量方面的研究还未见报道, 本实验克隆了大肠杆菌的 IPP 异构酶基因(idi)并将其转入小球藻中, 发现叶黄素的含量和产量得到提高图3 PCR 扩增鉴定转化子 Fig.3 PCR amplification to identify transformants a. PCR 扩增 idi 基因; b. PCR 扩增 NPT II 基因图5 Fig.5 图4 小球藻转化子和野生型的叶黄素含量 Lutein content of C. vulgaris transformants and wide type strain 小球藻转化子和野生型的生物量 Fig.4 Biomass of C. vulgaris transformants and wide type strain 注数据柱上方不同字母表示差异显著(P<0.05) 图 5 图 6 同图6 Fig.6 小球藻转化子和野生型的叶黄素产量 Lutein yield of C. vulgaris transformants and wide type strain

5 1 : (Agrobacterium tumefaciens) (Chlorella vulgaris) 163, idi, G418 PCR,,, 0.84mg/g, 30.95%, 1.98mg/L, 36.77%, idi, idi,,, T-DNA,, Cheng (2012), (, 2010),,,,, , 16(1): 84 86,,, , 31(01): ,,, IPP., 11(1): Anila N, Chandrashekar Arun, Ravishankar G A et al, Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil. European Journal of Phycology, 46(1): Bundock P, den Dulk-Ras A, Beijersbergen A et al, Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO Journal, 14(13): Carpentier S, Knaus M, Suh M, Associations between lutein, zeaxanthin, and age-related macular degeneration: an overview. Critical Reviews in Food Science and Nutrition, 49(4): Cha T S, Chen C F, Yee W et al, Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp.. Jounal of Microbiological Methods, 84(3): Cha T S, Yee W, Aziz A, Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World Journal of Microbiology and Biotechnology, 28(4): Chen Y, Wang Y, Sun Y et al, Highly efficient expression of rabbit neutrophil peptide-1 gene in Chlorella ellipsoidea cells. Current Genetics, 39(5 6): Cheng R B, Ma R J, Li K et al, Agrobacterium tumefaciens mediated transformation of marine microalgae Schizochytrium. Microbiological Research, 167(3): Chow K-C, Tung W L, Electrotransformation of Chlorella vulgaris. Plant Cell Reports, 18(9): Cordero B F, Couso I, Leon R et al, Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Applied Microbiology and Biotechnology, 91(2): Dawson H N, Burlingame R, Cannons A C, Stable transformation of Chlorella: rescue of nitrate reductase-deficient mutants with the nitrate reductase gene. Current Microbiology, 35(6): Dwyer J H, Navab M, Dwyer K M et al, Oxygenated carotenoid lutein and progression of early atherosclerosis: the Los Angeles atherosclerosis study. Circulation, 103(24): Fernandez-Sevilla J M, Acien Fernandez F G, Molina Grima E, Biotechnological production of lutein and its applications. Applied Microbiology and Biotechnology, 86(1): Hooykaas P J J, Roobol C, Schilperoort R A, Regulation of the transfer of Ti-plasmids of Agrobacterium tumefaciens. Journal of General Microbiology, 110(1): Huang J C, Wang Y, Sandmann G et al, Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta). Applied Microbiology and Biotechnology, 71(4): Kathiresan S, Chandrashekar A, Ravishankar G A et al, Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). Journal of Phycology, 45(3): Kim D H, Kim Y T, Cho J J et al, Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoide. Marine Biotechnology, 4(1): Kumar S V, Misquitta R W, Reddy V S et al, Genetic transformation of the green alga-chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Science, 166(3): Shen W J, Forde B G, Efficient transformation of Agro-

6 bacterium spp. by high voltage electroporation. Nucleic Acids Research, 17(20): Wang C H, Wang Y Y, Su Q et al, Transient expression of the GUS gene in a unicellular marine green alga, Chlorella sp. MACC/C95, via electroporation. Biotechnology and Bioprocess Engineering, 12(2): THE RESEARCH OF AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION OF GENE ENGINEERING CHLORELLA VULGARIS RICH IN LUTEIN MA Rui-Juan, LIN Xiang-Zhi (Engineering Research Center of Marine Biological Resource Comprehensive Utilization, Third Institute of Oceanography, State Oceanic Administration, Xiamen, ) Abstract In this study, we used gene clone technology to construct binary vector pcambia2301-idi, and transferred it into Agrobacterium tumefaciens LBA4404 by electroporation. The T-DNA region of pcambia2301-idi was introduced into Chlorella vulgaris by A. tumefaciens-mediated transformation (ATMT), using G418 resistance gene (NPT ) as selective marker which can be used to screen positive clones. The result of PCR showed that idi gene and NPT gene were integrated into the genome of C. vulgaris. Further analysis found that the majority of the transformants displayed similar biomass compared with the wild type strain. While the highest lutein content of transformants reached 0.84mg/g, increasing by 30.95% compared with that of the wild type strain. And the result of analyzing lutein yield in the algal culture demonstrated that the highest lutein yield of transformants reached 1.98mg/L, increasing by 36.77% compared with that of the wild type strain. Key words Chlorella vulgaris, Agrobacterium tumefaciens, Genetic transformation, idi gene, Lutein

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林学园艺中国农学通报 2015,31(10):153-157 Chinese Agricultural Science Bulletin 蚬壳花椒资源繁育与有效成分研究进展杨辉李猛王平孙吉康代明龙中南林业科技大学生命科学与技术学院长沙 410004 摘要总结了蚬壳花椒的一般特性及生长发育特征地理分布差异组织培养扦插嫁接有性繁育遗传多样性分析药用成分的种类有效成分的分离纯化方法