摘要
自1968年蓝藻被证实具有遗传重组现象开始,越来越多的人致力于蓝藻基因工程研究。随着蓝藻基因工程的发展,有一批外源基因被导入到蓝藻中表达。主要涉及环境治理、代谢调控、营养保健品和基因工程药物表达等应用领域。但是,外源基因表达效率低下的瓶颈制约了蓝藻基因工程的发展,无法实现规模化生产应用。
影响基因表达的因素有很多。如:基因拷贝数、密码子偏好性、启动子、终止子、反义RNA技术以及蛋白表达策略等。本研究试图从选择启动子、改变SD序列入手,对改进蓝藻外源基因表达系统和提高外源基因表达效率做一些探讨。
集胞藻6803(Synechocystis sp. strain PCC6803)是一种单细胞蓝藻,具有生长速度快、培养条件简单、不产生毒素、细胞结构简单、遗传背景清楚、方便分子操作等的特点,在特殊培养条件下还可以进行异养生长,非常适合于利用光合生物反应器大规模生产。因此,集胞藻6803是种很好的蓝藻基因工程受体。
本研究选择集胞藻6803染色体DNA中cupA基因及其下游相连的两个片段分别作为上游整合平台(up)和下游整合平台(down),在二者间插入诱导型高效启动子、egfp(增强型绿色荧光蛋白基因)报告基因、卡那霉素抗性筛选标记,构建得到集胞藻6803同源重组双交换整合平台。
在整合平台的基础上,通过更换启动子和进行SD序列距离修饰,共得到6个带启动子和1个不带启动子的转化载体。把转化载体分别自然转化到野生型集胞藻6803中,经卡那霉素筛选得到7个转基因藻。它们分别含有能够被红光诱导的Pcpcβ(转pK-Pcpcβ集胞藻6803和转pK-Pcpcβ2集胞藻6803)、被温度诱导的PgroESL(转pK-PgroESL集胞藻6803和转pK-PgroESL2集胞藻6803)、被光照诱导的PpsbA(转pK-PpsbA集胞藻6803和转pK-PpsbA2集胞藻6803)和1个不带启动子的转pK-P0集胞藻6803。
根据启动子的不同诱导特性,分别设计梯度诱导条件诱导EGFP表达,通过检测EGFP(增强型绿色荧光蛋白)荧光检测分析。EGFP在488nm光激发下会辐射绿色荧光,利用激光共聚焦显微镜对藻细胞直接进行荧光活性检测。通过比较EGFP的检测结果,判断各转基因藻外源基因表达系统的表达效率。
本实验主要结果或结论:1.构建得到6个带启动子的转化载体;2.构建得到1个不带启动子的转化载体;3.通过自然转化获得了7个转基因藻株;4. PgroESL具有温度诱导增强表达作用;5. SD序列修饰具有上调表达作用。
Since 1968 genetic recombinant phenomenon was found in cyanobacteria, more and more people committed to the cyanobacteria genetic engineering. With the development of cyanobacteria genetic engineering, many exogenous genes were introduced to cyanobacteria. It mainly involved environmental governance, metabolic, nutrition and health products, genetic engineering drugs,and so on. However, the inefficient expression for exogenous gene restricted the development and application of cyanobacteria genetic engineering.
There are many factors affect gene expression in cyanobacteria. Such as, gene copy number, genetic code preference, promoter, terminator, antisense RNA technology and protein expression strategies. In this study, we choose different promoter and modify SD sequence, in order to improve the exogenous gene expression system of cyanobacteria and increase it’s expression efficiency.
Synechocystis sp. strain PCC6803 is an unicellular cyanobacteria. It has faster growth rate and need simple culture condition. It’s cell structure is simple and genetic background is clear. It is facilitate operation and able to grow by either autotrophically or heterotrophically in special culture condition. It is an ideal biology to produce exogenous protein in large-scale bioreactor photosynthetic. Therefore, Synechocystis sp. strain PCC6803 is a very good receptor for cyanobacteria gene engineering.
We choosed a fragment close with 3’terminal from cupA gene of Synechocystis sp. strain PCC6803 as the upstream integration platform(up) and another fragment linked to cupA gene as the downstream integration platform(down), and inserted efficient inducible promoter, egfp(enhanced green fluorescent protein gene), kanamycin resistance marker, constructed a homologous recombination double exchange integration platform.
On the basis of the integration platform, we replaced the promoter and modified SD sequence, and gained six transformation vectors with promoter and one transformation vector with no promoter. The transformation vectors were transformed into the wild-type Synechocystis sp. strain PCC6803 cell. We gained seven transgenic cyanobacteria after the kanamycin screening. They contain the red-induced Pcpcβ(Transgenic pk-PcpcβSynechocystis PCC6803 and Transgenic pk-Pcpcβ2 Synechocystis PCC6803), the temperature-induced PgroESL(Transgenic pk-PgroESL Synechocystis PCC6803 and Transgenic pk-PgroESL2 Synechocystis PCC6803), the light-induced PpsbA(Transgenic pk-PpsbA Synechocystis PCC6803 and Transgenic pk-PpsbA2 Synechocystis PCC6803) and a non-promoter (Transgenic pk-P0 Synechocystis PCC6803).
According to the promoter’s features, we designed gradient induced conditions to induce EGFP(enhanced green fluorescent protein) expression, and analysised expression efficiency by detecting EGFP fluorescence activity. Under the 488 nm excitation light, EGFP will eradiate green fluorescent. And the fluorescent from the active algal cells can be detected by using laser scanning confocal microscope directly. By comparing the test results of EGFP, we can judge the exogenous gene expression efficiency of the transgenic cyanobacteria.
The main experimental results or conclusions including, 1)constructed six transformation vectors with promoter; 2)constructed one non-promoter transformation vector; 3)obtained seven transgenic cyanobacterium through natural transformation; 4)the PgroESL can increased the expression of EGFP after temperature-induced; 5)SD sequence modified can also up-regulated the expression of EGFP.
引文
[1] 楼士林,杨盛昌,龙敏南,等.基因工程[M].北京:科学技术出版社, 2002.355~389.
[2] 胡鸿钧,魏印心.中国淡水藻类:系统、分类及生态[M].北京:科学技术出版社, 2006.23.
[3] 施定基,张超,李世明,等.蓝藻与植物叶绿体光合系统基因的生物信息学研究.遗传学报[J]. 2004,31(6):627~633.
[4] 秦松 , 曾呈奎 . 藻类分子遗传学和基因工程研究的现状与展望 (I). 海洋科学 [J]. 1993,(1):34~37.
[5] 江红霞,郑怡.微藻的药用、保健价值及研究开发现状(综述).亚热带植物科学[J]. 2003,32(1):68~72.
[6] 敖宗华 , 汤晓智 , 刘萍 , 等 . 蓝藻中生物活性物质的研究概况 . 药物生物技术 [J]. 2001,8(5):296~300.
[7] Kaneko T, Sato S, Kotani H,et al.Sequence Analysis of the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence Determination of the Entire Genome and Assignment of Potential Protein-coding Regions. DNA Research[J]. 1996,3(3):109~136.
[8] Kaneko T, Sato S, Kotani H,et al.Sequence Analysis of the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence Determination of the Entire Genome and Assignment of Potential Protein-coding Regions(supllement). DNA Research[J]. 1996,3(3):185~209.
[9] Porter RD.Transformation in cyanobateria. Critical Reviews in Micribiology[J]. 1986,13(2):111~132.
[10] Billi D, Friedmann EI, Helm RF, et al. Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis. J Bacteriol[J]. 2001, 183(7):2298~2305.
[11] Koksharova OA, Wolk CP. Genetic tools for cyanobacteria. Appl Microbiol Biotechnol[J]. 2002, 58(2): 123~137.
[12] Scholz P, Haring V, Wittmann-Liebold B, et al. Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene[J]. 1989,75(2): 271~288.
[13] Bagdasarian M, Lurz R, Ruckert B, et al. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene[J]. 1981, 16(1-3):237~247.
[14] Sode K, Tatara M, Takeyama H, et al. Conjugative gene transfer in marine cyanobacteria: Synechococcus sp. Synechocystis sp. and Pseudanabaena sp. Appl Microbiol Biotechnol[J]. 1992, 37(3):369~373.
[15] Marraccini P, Bulteau S, Cassier-Chauvat C, et al. A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. Plant Mol Biol[J]. 1993, 23(4):905~909.
[16] Thiel T, Poo H. Transformation of a filamentous cyanobacterium by electroporation. JBacteriology[J]. 1989, 171(10):5743~5746.
[17] Muhlenhoff U, Chauvat F. Gene transfer and manipulation in the thermophilic cyanobacterium Synechococcus elongatus. Mol Gen Genet[J]. 1996, 252(1-2):93~100.
[18] Williams JGK. Construction of specific mutations in the photosystem II photosynthetic reaction center by genetic engineering methods in the cyanobacterium Synechocystis 6803. Meth. Enzymol[J]. 1988,167:766~778.
[19] Kuhlemeier1 CJ, Borrias1 WE, Van den Hondel1 CAMJJ,et al. Vectors for cloning in cyanobacteria: Construction and characterization of two recombinant plasmids capable of transformation to Escherichia coli K12 and Anacystis nidulans R2. Molecular and General Genetics MGG[J]. 1981,184 (2):249~254.
[20] Sherman LA, Van de putte PJ. Construction of a hybride plasmid capable of replication into bacteria:Synechococcus sp. Synechocystis sp. and Psedonabaena sp. Appl Microbiol Biotechnol[J]. 1982, 37:369~373.
[21] Wolk CP, Vonshak A, Kehoe P, et al. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proceedings of the National Academy of Sciences[J]. 1984, 81(5): 1561~1565.
[22] Flores E, Wolk CP. Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation. J Bacteriol[J]. 1985, 62:1399~1341.
[23] Golden SS. Mutagenesis of cyanobacteria by classical and gene-transfer-based methods. Methods Enzymol[J]. 1988, 167: 717~727.
[24] Essich E, Stevens SEL, Porter RD. Chromosomal transformation in the cyanobacterium Agmenellum guadruplicatum. J Bacteriol[J]. 1990, 172: 1916~1922.
[25] 魏兰珍,马为民,王全喜,等.蓝藻基因转移系统的选择与建立,中国生物工程杂志[J]. 2004,24(1):18~22.
[26] 陈颖,赵世民,孙勇如.藻类基因工程研究进展及展望.海洋科学[J]. 1997,(4):13~16.
[27] 秦松,严小军,曾呈奎.藻类分子生物技术两年评—基因工程及其上游—分子遗传学,海洋与湖沼[J]. 1996.27(1):103~111.
[28] 闵红涛,王业勤.蓝藻的一种人工转化系统研究.水生生物学报[J]. 1995,19(3):227~233.
[29] Koksharova OA, Wolk CP. Genetic tools for cyanobacteria. Applied Microbiology and Biotechnology[J]. 2002,58:123~137.
[30] Elhai J. Genetic techniques appropriate for biotechnological exploitation of cyanobacteria. Appl Phycol[J]. 1994, 6:177~186.
[31] Padan E, Shilo M. Cyanophages-viruses attacking blue-green algae. Bacteriol Rev[J]. 1973, 37(3): 343~370.
[32] Bergh O, Borsheim KY, Bratbak G, et al. High abundance of viruses found in aquatic environments. Nature[J]. 1989, 340(6233): 467~468.
[33] Hambly E, Tetart F, Desplats C, et al.2001. A conserved genetic module that encodes themajor virion components in both the coliphage T4 and the marine cyanophage S-PM2. Proc. Natl. Acad. Sci. USA[J].1998:11411~11416.
[34] Bazin MJ. Sexuality in a Blue–Green Alga: Genetic Recombination in Anacystis nidulans. Nature[J]. 1968,218:282 ~283.
[35] Shestakov SV, Khyen NT. Evidence for Genetic Transformation in Blue-Green Alga Anacystis nidulans. Melec. Gen Genetics[J]. 1970,107(4):372~375.
[36] Stevens SE, Porter RD. Transformation in Agmenellum quadruplicatum. Proceedings of the National Academy of Sciences[J]. 1980, 77: 6052~6056.
[37] Tandeau MN, Borrias WE, Kuhlemeier CJ, et al. A new approach for molecular cloning in cyanobacteria:cloning of an Anacystis nidulans met gene using a Tn901-induced mutant. Gene[J]. 1982,20(1):111~119.
[38] 孙军,金苹,徐旭东,等.小鼠金属硫蛋白—I cDNA 在蓝藻中的克隆和表达.生物工程进展[J]. 1994,14(6):39~42.
[39] 宋凌云,施定基,宁叶,等.用同源重组法将人肝金属硫蛋白突变体 ββ 基因整合在集胞藻6803 中表达.植物学报[J]. 2001,43(4):399~404.
[40] 周杰,罗娜,宁叶,等.通过同源重组在聚球藻 7002 中表达小鼠金属硫蛋白—Ⅰ的初步研究.生物化学与生物物理进展[J]. 2002,29 (1):149~153.
[41] Suzuki T, Miyake M, Tokiwa Y, et al. A recombinant cyanobacterium that accumulates poly-(hydroxybutyrate). Biotechnology Letters[J]. 1996,18(9):1047~1050.
[42] Sangthongpitag K, Penfold RJ, Delaney SF, et al. Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301. Applied Microbiology and Biotechnology[J]. 1997,47(4): 379~384.
[43] Angsuthanasombat C, Panyim S. Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6. Appl Environ Microbiol[J]. 1989,55(9):2428~2430.
[44] Murphy RC, Stevens SE.Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity. Appl Environ Microbiol[J]. 1992,58(5):1650~1655.
[45] Kawata Y, Yamano N, Kojima H, et al. Expression of salmon growth hormone in the cyanobacterium Agmenellum quadruplicatum. Biotechnology Letters[J]. 1991,13(12): 851~856.
[46] 张春莉,施定基,黄倢,等.白斑综合症病毒(WSSV)囊膜蛋白 VP28 基因的克隆及在蓝藻中表达载体的构建.海洋科学[J]. 2003,27(2):72~76.
[47] 冯燕,陈晓,施定基,等.水稻胞质 FBA 基因在鱼腥藻 7120 中的表达及其对光合作用的调控.植物研究[J]. 2006,26(6):691~698.
[48] 陈晓,谭玮,冯燕,等.水稻 cFBA 和菠菜 cpTPI 串联基因在鱼腥藻 7120 中的表达及其对光合作用的影响.上海师范大学学报(自然科学版) [J]. 2006,35(4):75~81.
[49] 唐功利,杨春松,鲍建绍,等.丙糖磷酸异构酶、果糖-1,6-二磷酸醛缩酶及果糖-1,6-二磷酸酶的共表达.生物化学与生物物理学报[J]. 2001,33(1):131~136.
[50] Wada H, Combos Z, Murata N. Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature[J]. 1990,347:200~203.
[51] Reddy AS, Nuccio ML,Gross LM. Isolation of a A6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120. Plant Molecular Biology[J]. 1993,27: 293~300.
[52] Takeshima Y, Takatsugu N, Sugiura M. High-Level Expression of Human Superoxide Dismutase in the Cyanobacterium Anacystis nidulans 6301. Proceedings of the National Academy of Sciences[J]. 1994,91:9685~9689.
[53] Chemeresiuk NN,Elanskaia IV. Cloning and expression of the B1 hordein gene of barley (Hordeum vulgare L.) in cells of the cyanobacterium Synechocystis sp. PCC6803 and Escherichia coli. Genetika[J]. 1994,30(9):1141~1145.
[54] Harker M,Hirschberg J. Biosynthesis of ketocarotenoids in transgenic cyanobacteria expressing the algal gene for beta-C-4-oxygenase, crtO. FEBS Letts[J]. 1997,404(2-3):129~134.
[55] 刘凤龙,张宏斌,施定基,等.人肿瘤坏死因子 α 基因穿梭表达载体的构建和在鱼腥藻 7120中的表达.中国科学(C 辑) [J]. 1999,29(2):217~224.
[56] 戴溦,施定基,张卉,等.人表皮生长因子(hEGF)基因在蓝藻中的表达.植物学报[J]. 2001,43(12):1260~1264.
[57] 罗娜,宁叶,施定基,等.人尿激酶原基因在聚球藻 7002 中的克隆和表达. 植物学报[J]. 2000,42(9):931~935.
[58] 章军,宋新强,徐虹,等.利用同源重组质粒 pUTK 转化蓝藻 Synechococcus sp. PCC7942 及胸腺素 α1 的表达. 海洋科学[J]. 2001,25(6):1~3.
[59] 陈翠丽,施定基.转人粒细胞集落刺激因子(hG-CSF)基因的鱼腥藻的构建.北京联合大学学报,2005,19(3):60~63.
[60] 魏兰珍,金锐,马为民,等.hGM-CSF 基因穿梭表达载体的构建及其在鱼腥藻 7120 中的克隆. 植物研究[J]. 2005,25(4):436~440.
[61] Hockney RC. Recent developments in heterologous protein production in Escherichia coli. Trends biotechnol[J]. 1994, 12: 456~463.
[62] Buckholz RG.. Yeast system for the expression of heterologous gene products. Curr Opin Biotechnol[J]. 1993, 4:538~542.
[63] Luckow VA. Baculovirus systems for the expression of human gene products. Curr Opin Biotechnol[J]. 1993, 4:564~572.
[64] Levinson AD. Expression of heterogous gene in mammalian cells[J]. Meth Enzymol[J]. 1990, 185: 485~487.
[65] 王 业 勤 , 徐 旭 东 , 黎 尚 毫 . 蓝 藻 分 子 遗 传 学 十 年 研 究 进 展 . 水 生 生 物 学 报 [J]. 1991,15(4):356~367.
[66] Golden SS, Sherman LA. Optimal condition for genetic transformation of cyanobacterium Anacystis nidulans R2. J Bacteriol[J]. 1984, 158: 36~42.
[67] Marsac NT, Torre F, Szulmaister J. Expression of the larvicidal gene of Bacillus sphoericus 1593M in the cyanobacterium Anacystis nidulans R2. Mol.Gen.Genet.[J]. 1987, 209: 296~298.
[68] 施定基.用转基因蓝藻制备重组药物的发展与展望.2000,中国药物生物技术学术研讨会.
[69] 肖生科,王磊,陈毓荃.提高外源基因在巴斯德毕赤酵母中表达量的研究进展.生物技术通报[J]. 2004,(2):23~26.
[70] 刘丽,谢宝树.利用双顺反子结构提高人 GM—CSF 在大肠杆菌中的表达水平.白求恩医科大学学报[J]. 1996,22(4):336~338.
[71] Aoyama K, Miyake M, Yamada J, et al. Application of vector pKE4-9 carrying a strong promoter to the expression of foreign proteins in Synechococcus PCC7942. Journal of Marine Biotechnology[J]. 1996,4(1):64~67.
[72] Wilbur H,Campbell,G. Gowri. Codon Usage in Higher Plants, Green Algae, and Cyanobacteria. Plant Physiology[J]. 1990,92:1~11.
[73] Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev[J]. 1996,60(3):512~538.
[74] Wei Lanzhen, Ma Weimin, Shi Dingji, et al. Modification of the N-Terminal Nucleotide Sequence of Mature hGM-CSF Results in High Expression in the Foreign Host, Anabaena sp. Strain PCC 7120. Journal of Applied Phycology[J]. 2006,18(2):153~159.
[75] 张 春 晓 , 王 文 棋 , 蒋 湘 宁 , 等 . 植 物 基 因 启 动 子 研 究 进 展 . 植 物 学 报 [J]. 2004,31(13):1455~1464.
[76] 高宏,唐蜻,徐旭东.集胞藻 PCC6803 铜离子诱导平台的构建.水生生物学报[J]. 2007,31(2):240~244.
[77] Bissati KEl, Kirilovsky D. Regulation of psbA and psaE Expression by Light Quality in Synechocystis Species PCC 6803. A Redox Control Mechanism. Plant Physiol[J]. 2001,125:1988~2000.
[78] 赵军,范云六.水稻种子醇溶蛋白 4a 基因的表达受串联复合启动子调控.中国科学(C 辑)[J]. 1996,26(2):156~163.
[79] Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem[J]. 1988,57:199~233.
[80] Gold L, Stormo GD. High-level translation initiation. Methods Enzymol[J]. 1990, 185:89~93.
[81] 李宏,杨体强,达赖,等.大肠杆菌 SD 序列与基因表达水平的关系.内蒙古大学学报(自然科学版)[J]. 1998,29(2):172~176.
[82] 秦京东,施定基,徐旭东,等.在鱼腥藻 7120 中建立反义 glnA 系统.植物生理学报[J]. 1998,24(3):225~232.
[83] 王彪 , 武天龙 . 提高外源基因在植物体内表达的策略 . 热带亚热带植物学报 [J]. 2005,13(1):80~84.
[84] Spence E, Sarcina M, Ray N, et al. Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803. Molecular Microbiology[J]. 2003,48(6):1481~1489.
[85] 谢磊,孙建波,张世清,等.大肠杆菌表达系统及其研究进展.华南热带农业大学学报[J]. 2004,10(2):16~20.
[86] 周杰,郝福英,施定基,等.小鼠金属硫蛋白-I 在鱼腥藻 7120 中的融合表达及其纯化(英文). 植物学报(英文版)[J]. 2003,45(1):98~101.
[87] 陶天申,杨瑞馥,东秀珠.原核生物系统学[M].北京:化学工业出版社,2007.323~330.
[88] Poter RD. Transformation in cyanobacteria. CRC Critical reviews in microbiology[J]. 1986, 13(2):111~132.
[89] Blattner FR, Plunkett G, Bloch CA, et al. The Complete Genome Sequence of Escherichia coli K-12. Science [J]. 1997,277(5331):1453~1462.
[90] Morie JG.. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry[J]. 1974,13(12):2656~2562.
[91] Prasher DC, Eckenrode VK, Wade WW, et a1. Primary structure of the Aequorea Victoria green fluorescent protein. Gene[J]. 1992,111(2):229~233.
[92] 金鹰.绿色荧光蛋白(GFP)研究进展.激光生物学报[J]. 1999,8(3):228~233.
[93] Yang F, Moss LG., Phillips GN. The molecular structure of green fluorescent protein. Nature Biotechnology[J]. 1996, 14(10):1246~1251.
[94] Cormack BP, Valdivia R, Falkow S. FACS-optimized mutants of the green fluorescent protein. Gene[J]. 1996,173:33~38.
[95] Elhai J. Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiology Letters[J]. 1993,114:179~184.
[96] Pilot TJ, Fox JL. Cloning and Sequencing of the Genes Encoding the and ? Subunits of C-phycocyanin from the Cyanobacterium Agmenellum quadruplicatum. Proceedings of the National Academy of Sciences[J]. 1984,81(22):6983~6987.
[97] Webb R, Reddy KJ, Sherman LA. Regulation and sequence of the Synechococcus sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin. J Bacteriol[J]. 1990,172(9): 5079~5088.
[98] Takeshima Y, Takatsugu N, Sugiura M. High-Level Expression of Human Superoxide Dismutase in the Cyanobacterium Anacystis nidulans 6301. Proceedings of the National Academy of Sciences[J]. 1991:9685~9689.
[99] 吴乃虎.基因工程原理[M].(第二版).北京:科学出版社, 2001. 185~192.
[100] Rippka R. Isolation and purification of cyanobacteria. Methods Enzyme[J]. 1988,167:3~27.
[101] J. 萨姆布鲁克, D.W. 拉塞尔.分子克隆实验指南(上、下册)[M].(第三版).黄培堂等译. 北京:科学出版社,2002. 2~146.
[102] C.W.迪芬巴赫, G.S.德维克斯勒. PCR 实验技术指南[M].黄培堂, 俞炜源, 陈添弥, 等译.北京:科学出版社,1998. 380~415.
[103] 李湘萍,徐慰倬,李宁.动物基因敲除研究的现状与展望.遗传[J]. 2003,25(1):81~88.
[104] Hinnen A, Hicks JB, Fink GR. Transformation of yeast. Proceedings of the National Academy of Sciences[J]. 1978,78:1929~1933.
[105] Smithies O, Gregg RG, Boggs SS, et al. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination . Nature[J]. 1985,317:230~234.
[106] 高翔.基因靶向改造技术的发展及其意义—解读 2007 年诺贝尔生理学或医学奖.生物化学与生物物理进展[J]. 2007,34(10):1009~1011.
[107] 田三德,孙鹏.基因打靶技术及其应用.陕西科技大学学报[J]. 2003,21(4)94~98.
[108] Shulman MJ, Nissen L, Collins C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol cell Biol[J]. 1990,10:4466~4472.
[109] Thomas KR, Deng C, Capecchi MR. High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol Cell Biol[J]. 1992,12(7):2919~2923.
[110] 刘默芳,王恩多.绿色荧光蛋白.生物化学与生物物理进展[J]. 2000,27(3):238~243.
[111] 罗文新,陈敏,程通,等.橙色荧光蛋白—绿色荧光蛋白 GFPxm 的改造.生物工程学报[J]. 2003,(19):156~62.
[112] 黄国存,朱生伟,董越梅,等.绿色荧光蛋白及其在植物研究中的应用,植物学通报[J]. 1998,15(5):24~30.
[113] Goloubinoff P, Gatenby AA, Lorimer GH. GroE heat-schock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escharichia coli. Nature[J]. 1989,337:44~47.
[114] Creighton TE. Unfolding protein folding. Nature[J]. 1991,352:17~18.