葡萄硫氧还蛋白基因(VvTrx)的克隆、表达和原核表达

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Abstract Thioredoxin (Trx) is a kind of highly conservative and low molecular weight proteins, which widely exists in plants, animals, bacteria and yeasts. It maintains the redox equilibrium of organisms, which regulates and controls biological signal transduction and other functions. This study cloned VvTrx gene from grape (Vitis vinifera) gDNA sequence (GenBank No. EU280166) of 989 bp and cDNA sequence (GenBank No. EU280164) of 561 bp and the open reading frame (ORF) was 381 bp, encoding 126 amino acids. The multiple sequence alignment and phyogenetix tree showed that the active site of VvTrx gene in Thompson Seedless (Vitis vinifera cv.) was WCIPS and belonged to the subgroup Ⅳ in Trx-h. Analysis of quantitative Real-time PCR(qRT-PCR) showed that, in different periods of the ovule development of both Thompson Seedless and Pinot Noir, the expression of VvTrx gene reached the highest level in 40 d after flowering. Besides, the difference between them was extremely significant (P＜0.001) and the expression of Thompson Seedless was about 5 times of Pinot Noir. After that, the VvTrx gene was cloned into the prokaryotic expression vector of pET-32a(+) to construct pET-32a(+)/VvTrx, and a 30 kD solubility fusion protein expressed in the Escherichia coli BL21 through isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The study showed that the VvTrx gene had a certain relation to the process of seedless grape ovules abortion, which provides the research directions to the molecular mechanism of VvTrx gene in seedless grape embryo abortion process and the breeding of seedless grape.

Key words： Grape Thioredoxin(Trx) Cloning Expression analysis Fusion expression

Received: 16 February 2015 Published: 09 July 2015

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	ZHANG Xiao-Ying
	TANG Yu-Jin
	YU Gang
	YU Ti-Jin
	ZHANG Zhao-Hong

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ZHANG Xiao-Ying,TANG Yu-Jin,YU Gang, et al. Cloning, Expression and Fusion Expression of Thioredoxin Gene (Trx) in Grape (Vitis vinifera)[J]. , 2015, 23(9): 1131-1140.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2015/V23/I9/1131

李桂荣, 王跃进, 唐冬梅, 等. 2001. 无核白葡萄胚挽救育种技术研究[J]. 西北植物学报, 21(3): 432-436.(Li G R, Wang Y J, Tang D M, et al. 2001. The studies on embryo rescue techniques of‘Thompson Seedless’grape [J].Acta Botanica Boreali-Occidentalia Sinica, 21(3): 432-436.)
王跃进, Lamikanra O, Schell L, 等. 1997. 用 RAPD 分析鉴定葡萄属远缘杂种[J]. 西北农业大学学报, 25(3): 16-20.（Wang Y J, Lamikanra O, Schell L, et al.1997. Identification of Hybrids Derived from Vitis rotundifolia×Vitis vinifera by RAPD Analysis[J]. Journal of Northwest Sci-Tech University of Agriculture and Foresty, 25(3): 16-20.）
张今今, 王跃进, 王西平, 等. 2003. 葡萄总RNA提取方法的研究[J]. 果树学报, 20(3) : 178-181. (Zhang J J, Wang Y J, Wang X P, et al. 2003. An Improved Method for Rapidly Extracting Total RNA from Vitis [J]. Journal of Fruit Science, 20(3) : 178-181. )
Arnér, E. S., Holmgren, A. 2000. Physiological functions of thioredoxin and thioredoxin reductase[J]. European Journal of Biochemistry, 267 (20):6102-6109.
Balmer, Y., Vensel, W. H., Tanaka, C. K., Hurkman, W. J., Gelhaye, E., Rouhier, N., Jacquot, J.-P., Manieri, W., Schürmann, P., Droux, M. 2004. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria[J]. Proceedings of the National Academy of Sciences of the United States of America, 101 (8):2642-2647.
Bashandy, T., Guilleminot, J., Vernoux, T., Caparros-Ruiz, D., Ljung, K., Meyer, Y., Reichheld, J.-P. 2010. Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling[J]. The Plant Cell Online, 22 (2):376-391.
Cheng, Z., Arscott, L. D., Ballou, D. P., Williams, C. H. 2007. The relationship of the redox potentials of thioredoxin and thioredoxin reductase from Drosophila melanogaster to the enzymatic mechanism: reduced thioredoxin is the reductant of glutathione in Drosophila[J]. Biochemistry, 46 (26):7875-7885.
de Dios Barajas-López, J., Serrato, A. J., Olmedilla, A., Chueca, A., Sahrawy, M. 2007. Localization in roots and flowers of pea chloroplastic thioredoxin f and thioredoxin m proteins reveals new roles in nonphotosynthetic organs[J]. Plant Physiology, 145 (3):946-960.
de Matos, A. L., Liu, J., McFadden, G., Esteves, P. J. 2013. Evolution and divergence of the mammalian SAMD9/SAMD9L gene family[J]. BMC evolutionary biology, 13 (1):121.
Domkin, V., Chabes, A. 2014. Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins[J]. Scientific reports, 4.
Fujino, G., Noguchi, T., Matsuzawa, A., Yamauchi, S., Saitoh, M., Takeda, K., Ichijo, H. 2007. Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1[J]. Molecular and cellular biology, 27 (23):8152-8163.
Gelhaye, E., Rouhier, N., Jacquot, J.-P. 2004. The thioredoxin h system of higher plants[J]. Plant Physiology and Biochemistry, 42 (4):265-271.
Gelhaye, E., Rouhier, N., Navrot, N., Jacquot, J. 2005. The plant thioredoxin system[J]. Cellular and Molecular Life Sciences CMLS, 62 (1):24-35.
Holmgren, A. 1989. Thioredoxin and glutaredoxin systems[J]. J Biol Chem, 264 (24):13963-13966.
Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., Gotoh, Y. 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways[J]. Science, 275 (5296):90-94.
Ishiwatari, Y., Honda, C., Kawashima, I., Nakamura, S.-i., Hirano, H., Mori, S., Fujiwara, T., Hayashi, H., Chino, M. 1995. Thioredoxin h is one of the major proteins in rice phloem sap[J]. Planta, 195 (3):456-463.
Jacquot, J. P., Rouhier, N., Gelhaye, E. 2002. Redox Control by Dithiol‐Disulfide Exchange in Plants[J]. Annals of the New York Academy of Sciences, 973 (1):508-519.
Juárez-Díaz, J. A., McClure, B., Vázquez-Santana, S., Guevara-García, A., León-Mejía, P., Márquez-Guzmán, J., Cruz-García, F. 2006. A novel thioredoxin h is secreted in Nicotiana alata and reduces S-RNase in vitro[J]. Journal of Biological Chemistry, 281 (6):3418-3424.
Kamikawa, R., Inagaki, Y., Hashimoto, T. 2014. Secondary loss of a cis-spliced intron during the divergence of Giardia intestinalis assemblages[J]. BMC research notes, 7 (1):413.
Kyriakis, J. M., Avruch, J. 2001. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[J]. Physiological reviews, 81 (2):807-869.
Laloi, C., Rayapuram, N., Chartier, Y., Grienenberger, J.-M., Bonnard, G., Meyer, Y. 2001. Identification and characterization of a mitochondrial thioredoxin system in plants[J]. Proceedings of the National Academy of Sciences, 98 (24):14144-14149.
Lynch, M. 2002. Intron evolution as a population-genetic process[J]. Proceedings of the National Academy of Sciences, 99 (9):6118-6123.
Meng, L., Wong, J. H., Feldman, L. J., Lemaux, P. G., Buchanan, B. B. 2010. A membrane-associated thioredoxin required for plant growth moves from cell to cell, suggestive of a role in intercellular communication[J]. Proceedings of the National Academy of Sciences, 107 (8):3900-3905.
Meyer, Y., Belin, C., Delorme-Hinoux, V., Reichheld, J.-P., Riondet, C. 2012. Thioredoxin and glutaredoxin systems in plants: molecular mechanisms, crosstalks, and functional significance[J]. Antioxidants & redox signaling, 17 (8):1124-1160.
Meyer, Y., Vignols, F., Reichheld, J. P. 2002. Classification of plant thioredoxins by sequence similarity and intron position[J]. Methods in enzymology, 347394-402.
Miranda‐Vizuete, A., Damdimopoulos, A. E., Pedrajas, J. R., Gustafsson, J. ?., Spyrou, G. 1999. Human mitochondrial thioredoxin reductase[J]. European Journal of Biochemistry, 261 (2):405-412.
Motohashi, K., Kondoh, A., Stumpp, M. T., Hisabori, T. 2001. Comprehensive survey of proteins targeted by chloroplast thioredoxin[J]. Proceedings of the National Academy of Sciences, 98 (20):11224-11229.
Ramming, D. W., Emershad, R. L., Tarailo, R. 2000. A stenospermocarpic, seedless Vitis vinifera× Vitis rotundifolia hybrid developed by embryo rescue[J]. HortScience, 35 (4):732-734.
Sahrawy, M., Hecht, V., Lopez-Jaramillo, J., Chueca, A., Chartier, Y., Meyer, Y. 1996. Intron position as an evolutionary marker of thioredoxins and thioredoxin domains[J]. Journal of molecular evolution, 42 (4):422-431.
Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., Ichijo, H. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1[J]. The EMBO journal, 17 (9):2596-2606.
Schürmann, P., Buchanan, B. B. 2008. The ferredoxin/thioredoxin system of oxygenic photosynthesis[J]. Antioxidants & redox signaling, 10 (7):1235-1274.
Serrato, A. J., Crespo, J. L., Florencio, F. J., Cejudo, F. J. 2001. Characterization of two thioredoxins h with predominant localization in the nucleus of aleurone and scutellum cells of germinating wheat seeds[J]. Plant molecular biology, 46 (3):361-371.
Serrato, A. J., Guilleminot, J., Meyer, Y., Vignols, F. 2008. AtCXXS: atypical members of the Arabidopsis thaliana thioredoxin h family with a remarkably high disulfide isomerase activity[J]. Physiologia plantarum, 133 (3):611-622.
Tonissen, K. F., Di Trapani, G. 2009. Thioredoxin system inhibitors as mediators of apoptosis for cancer therapy[J]. Molecular nutrition & food research, 53 (1):87-103.
Traverso, J. A., Micalella, C., Martinez, A., Brown, S. C., Satiat-Jeunema?tre, B., Meinnel, T., Giglione, C. 2013. Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study[J]. The Plant Cell, 25 (3):1056-1077.
Verdoucq, L., Vignols, F., Jacquot, J.-P., Chartier, Y., Meyer, Y. 1999. In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family[J]. Journal of Biological Chemistry, 274 (28):19714-19722.
Wang, J., Horiuchi, S. 1990. A histological study on the seedlessness in'Himrod Seedless' grape[J]. Journal of the Japanese Society for Horticultural Science, 59 (3):455-462.
Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., Hisabori, T. 2004. Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana[J]. Plant and Cell Physiology, 45 (1):18-27.
Yang, L., Wu, D., Wang, X., Cederbaum, A. I. 2011. Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1–JNK1 pathway in HepG2 cells[J]. Free Radical Biology and Medicine, 51 (1):185-196.
Yoo, M.-H., Carlson, B. A., Gladyshev, V. N., Hatfield, D. L. 2013. Abrogated thioredoxin system causes increased sensitivity to TNF-α-induced apoptosis via enrichment of p-ERK 1/2 in the nucleus[J]. PloS one, 8 (9):e71427.
Zhang, R., Al-Lamki, R., Bai, L., Streb, J. W., Miano, J. M., Bradley, J., Min, W. 2004. Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner[J]. Circulation research, 94 (11):1483-1491.