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| Cloning and Expression Analysis of Cyt b5 Gene Involved in Electron Transfer in Gossypium hirsuturm |
| LIU Jin-zhi1,2, SI Huai-jun1, ZHANG Ning1, WU Jia-he2 |
1. Life Science Institute of Technology, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory of Plant Genomic, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China |
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Abstract On the basis of salt-stress related EST library from Gossypium hirsuturm L. A partial sequence fragment of cytochrome b5 proteins (Cyt b5) encoding gene was obtained through homologous comparison with other plants. The Cyt b5 gene was isolated by the 3',5'-RACE technology from G. hirsuturm, named GhCyt b5. The full-length cDNA of GhCyt b5 is 810 bp, containing a 402 bp ORF which encodes 134 amino-acid peptide. The relative molecular weight of GhCyt b5 protein is 17 kDa. The coding sequence fragment was amplified by PCR and cloned into vector pET32a to generate the pET32a-GhCyt b5 expression vector, which was transformed into BL21(DE3) for expression of recombinant protein. On the base of optimization of different inducible conditions, the results showed that soluble GhCyt b5 protein under the 28℃ and 1 mmol/L IPTG conditions could be obtained. The expression product of GhCyt b5 was purified by Ni2+ affinity column and identified by SDS-PAGE. Using extracted cotton cell material as reaction medium for in vitro electron transfer analysis, it can be found the GhCyt b5 involved in the electron transfer system and accepted electron to become oxidation state from reducing state. Ultimately, the Cyt b5 gene for the first time from G. hirsuturm was cloned and its participating in electron transfer system was confirmed, which provide a base for further dissecting function in biotic and abiotic resistance.
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Received: 15 January 2013
Published: 25 April 2013
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[1] Jiang B G. Optimization of Agrobacterium mediated cotton transformation using shoot apices explants and quantitative trait loci analysis of yield and yield component traits in upland cotton. Shandong Agricultural University, Doctoral Dissertation, 2004.
[2] Reddy V V, Kupfer D, Caspi E. Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. Biological Chemistry, 1977, 252:2797-2801.
[3] Keyes S R, Alfano J A, Jansson I, et al. Rat liver microsomal elongation of fatty acids. Biological Chemistry, 1979, 254:7778-7784.
[4] Noshiro M, Omura T. Immunochemical study on the electron pathway from NADH to cytochrome P-450 of liver microsomes. Biological Chemistry, 1978, 83:61-77.
[5] Smith M A, Stobart A K, Shewry P R, et al. Tobacco cytochrome b5: cDNA isolation, expression analysis and in vitro protein targeting. Plant Molecular Biology, 1994, 25:527-537.
[6] Sayanova O, Smith M A, Lapinskas P, et al. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco. Plant Biology, 1997, 94:4211-4216.
[7] Fan R C, Peng C C, Xu Y H, et al. Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars. Plant Physiology, 2009, 150:1880-1901.
[8] Li Y, Li L L, Fan R C, et al. Arabidopsis sucrose transporter SUT4 interacts with cytochrome b5-2 to regulate seed germination in response to sucrose and glucose. Molecular Plant, 2012, 5(5):1029-1041.
[9] Fukuchi-Mizutani M, Mizutani M, Tanaka Y, et al. Microsomal electron transfer in higher plants: cloning and heterologous expression of NADH-Cytochrome b5 reductase from Arabidopsis. Plant Physiology, 1999, 119:353-362.
[10] 李晶,王亦学,郑德刚,等.一种简单高效提取棉花不同组织总RNA的方法.山西农业科学, 2009,37(5):17-19. Li J, Wang Y X, Zheng D G, et al. A simply and high efficiency method of total RNA isolation in upland cotton. Shanxi Agricultural Sciences, 2009, 37(5):17-19.
[11] McCoy G D, DeMareo G J, Biaglow J A. Influence of chronic ethanol consumption on hamster liver microsomal-dealkylase activities and cytochrome b5 content. Biochemical Pharmacology, 1985, 34:4263-4267.
[12] Kwata S, Sugiyama T, Seki K, et al. Stimulatory effect of cytochrome b5 induced by p-nitroanisole and diisopropyl l,3-dithiol-2-ylidenemalonate on rat liver microsomal drug hydroxylations. The Journal of Biochemistry, 1982, 92:305-313.
[13] Kearns E V, Keck P, Somerville C R. Primary structure of cytochrome b5 from cauliflower (Brassica oleracea L.) deduced from peptide and cDNA sequences. Plant Physiology, 1992, 99:1254-1257.
[14] Martsinkovskaya A I, Poghosyan Z P, Haralampidis K, et al. Temporal and spatial gene expression of cytochrome b5 during flower and fruit development in olives. Plant Molecular Biology, 1999, 40:79-90.
[15] 黄圣兵,宋 玮,林其谁. 大鼠电子传递黄素蛋白-泛醌氧化还原酶cDNA的克隆功能表达和细胞定位.中国科学生命科学,2005,35 (1):77-86. Huang S B, Song W, Ling Q S. Scientia Sinica Vitae, 2005, 35 (1):77-86.
[16] 廖丹,谢建平,王洪海.结核分枝杆菌膜蛋白的异源表达与纯化研究进展.微生物学报,2007,47(5):932-936. Liao D, Xie J P, Wang H H. The heterologous expression and purification of membrane protein from Mycobacterium tuberculosis. Acta Microbiologica Sinica, 2007, 47(5): 932-936.
[17] Nica B, Renato L. Both the outer mitochondrial membrane and the microsomal forms of cytochrome b5 reductase contain covalently bound myristic acid. The Journal of Biochemistry, 1990, 266:341-347.
[18] Smith M A, Napier J A, Stymne S, et al. Expression of a biologically active plant cytochrome b5 in Escherichia coli. The Journal of Biochemistry, 1994, 303: 73-79.
[19] Sobrado P, Goren M A, James D, et al. A protein structure initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. Protein Expr Purif, 2008,58(2):229-241.
[20] Yubisui T, Takabayashi T, Takahashi F, et al. Structure of a cDNA for ciona cytochrome b5 and the ubiquitous expression of mRNA in embryonic tissues. The Journal of Biochemistry, 2004,135:231-236. |
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