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高粱SbGABA-Ts基因的克隆、原核表达及纯化

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.05.015, PP. 93-99

Keywords: 高粱GABA-T,原核表达,纯化,信号肽

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Abstract:

植物中GABA(γ-氨基丁酸)代谢与植物生长发育、信号传递及逆境响应等过程密切相关,而参与GABA代谢的关键酶GABA-T(γ-氨基丁酸转氨酶)在重要农艺作物中的研究相对滞后。利用同源性分析在高粱基因组数据库中获得两个γ-氨基丁酸转氨酶(SbGABA-T)基因,RT-PCR方法进行基因克隆,并连入原核表达载体pET28a(+),转化E.coliBL21(DE3)进行基因异源表达分析。结果表明,包含SbGABA-T编码区全长的融合蛋白主要在包涵体中表达,而去除了SbGABA-TN端信号肽的融合蛋白以部分可溶的形式存在。进一步优化表达条件,IPTG浓度为1mmol/L时,16℃低温诱导18h,即可获得大量可溶性融合蛋白。用带His标签的镍柱对融合蛋白进行了纯化。

References

[1]  王海莲, 管延安, 张华文, 等. 高粱基因组学研究进展[J]. 基因组学与应用生物学, 2009(3):549-556.
[2]  Paterson AH, Bowers JE, Bruggmann R, et al. The Sorghum bicolor genome and the diversification of grasses[J]. Nature, 2009, 457(7229):551-556.
[3]  Bouché N, Fromm H, GABA in plants:just a metabolite?[J]. Trends in Plant Science, 2004, 9(3):110-115.
[4]  Clark SM, Di Leo R, Dhanoa PK, et al. Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis γ-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate[J]. Journal of Experimental Botany, 2009, 60(6):1743-1757.
[5]  Renault H, Roussel V, El Amrani A, et al. The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance[J]. BMC Plant Biology, 2010, 10(1):20.
[6]  Deleu C, Faes P, Niogret MF, et al. Effects of the inhibitor of the gamma-aminobutyrate-transaminase, vinyl-gamma-aminobutyrate, on development and nitrogen metabolism in Brassica napus seedl-ings[J]. Plant Physiology and Biochemistry, 2013, 64:60-69.
[7]  李立奇, 万瑛. 蛋白质的亚细胞定位预测研究进展[J]. 免疫学杂志, 2009(5):602-604.
[8]  Akihiro T, Koike S, Tani R, et al. Biochemical mechanism on GABA accumulation during fruit development in tomato[J]. Plant and Cell Physiology, 2008, 49(9):1378-1389.
[9]  Van Cauwenberghe OR, Shelp BJ, Biochemical characterization of partially purified gaba:pyruvate transaminase from Nicotiana tabacum[J]. Phytochemistry, 1999, 52(4):575-581.
[10]  Allan WL, Simpson JP, Clark SM, et al. γ-Hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress:putative regulation by redox balance and glyoxylate reductase isoforms[J]. Journal of Experimental Botany, 2008, 59(9):2555-2564.
[11]  Morishige DT, Klein PE, Hilley JL, et al. Digital genotyping of sorg-hum-a diverse plant species with a large repeat-rich genome[J]. BMC Genomics, 2013, 14(1):448.
[12]  Shelp BJ, Mullen RT, Waller JC. Compartmentation of GABA metabolism raises intriguing questions[J]. Trends in Plant Science, 2012, 17(2):57-59.
[13]  Shelp BJ, Bozzo GG, Zarei A, et al. Strategies and tools for studying the metabolism and function of γ-aminobutyrate in plants. II. Integrated analysis[J]. Botany, 2012, 90(9):781-793.
[14]  Fait A, Fromm H, Walter D, et al. Highway or byway:the metabolic role of the GABA shunt in plants[J]. Trends in Plant Science, 2008, 13(1):14-19.
[15]  Clark SM, Di Leo R, Van Cauwenberghe OR, et al. Subcellular localization and expression of multiple tomato γ-aminobutyrate transaminases that utilize both pyruvate and glyoxylate[J]. Journal of Experimental Botany, 2009, 60(11):3255-3267.
[16]  Shimajiri Y, Ozaki K, Kainou K, et al. Differential subcellular loca-lization, enzymatic properties and expression patterns of gamma-am-inobutyric acid transaminases(GABA-Ts)in rice(Oryza sativa)[J]. Journal of Plant Physiology, 2013, 170(2):196-201.
[17]  Koike S, Matsukura C, Takayama M, et al. Suppression of gamma-aminobutyric acid(GABA)transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato(Solanum lycopersicum L.)[J]. Plant and Cell Physiology, 2013, 54(5):793-807.
[18]  Palanivelu R, Brass L, Edlund AF, et al. Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels[J]. Cell, 2003, 114(1):47-59.
[19]  Patnaik PR. Investigation of induction effect on the steady state performance of a continuous fermentation for recombinant β-ga1actosidase[J]. Process Biochemistry, 2001, 36(11):1069-1074.
[20]  Shelp BJ, Bown AW, McLean MD. Metabolism and functions of gamma-aminobutyric acid[J]. Trends in Plant Science, 1999, 4(11):446-452.
[21]  Hyun TK, Eom SH, Jeun YC, et al. Identification of glutamate decarboxylases as a γ-aminobutyric acid(GABA)biosynthetic enzyme in soybean[J]. Industrial Crops and Products, 2013, 49:864-870.
[22]  Renault H. Fiat lux!:phylogeny and Bioinformatics shed light on GABA functions in plants[J]. Plant Signaling and Behavior, 2013, 8(6):e24274.

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