<em>GhCyt b5</em>基因的克隆及其蛋白参与电子传递功能的分析

中国生物工程杂志

2013, Vol. 33

Issue (4): 68-73

研究报告

GhCyt b5基因的克隆及其蛋白参与电子传递功能的分析

刘金芝^1,2, 司怀军¹, 张宁¹, 吴家和²

1. 甘肃农业大学生命科学技术学院兰州 730070;
2. 国家植物基因组学重点实验室中国科学院微生物研究所北京 100101

Cloning and Expression Analysis of Cyt b5 Gene Involved in Electron Transfer in Gossypium hirsuturm

LIU Jin-zhi^1,2, 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

全文: PDF(756 KB) HTML

摘要： 从陆地棉品种中棉所35盐胁迫EST文库中筛选到与其它植物高度同源的细胞色素b5蛋白(Cyt b5 )基因片段,利用RACE技术,获得Cyt b5 基因的cDNA序列, 命名为GhCyt b5,基因全长810bp,最大阅读框402 bp,编码由134 个氨基酸组成的蛋白质,分子量约为17kDa。将该基因的编码序列插入到原核表达载体pET32a中,构建重组质粒为pET32a-Cyt b5,对不同条件下进行蛋白诱导表达分析,发现在28℃和1 mmol/L IPTG条件下能够获得可溶性的GhCyt b5蛋白。利用Ni²⁺柱亲和层析纯化和SDS-PAGE鉴定分析表明获得重组蛋白为目的蛋白。通过提取棉花细胞物质为反应介质,进行体外电子传递功能分析,发现GhCyt b5能够从还原态变为氧化态,参与电子的传递功能。

关键词： 棉花; 细胞色素b5蛋白; 原核表达; 电子传递

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.

Key words: Gossypium hirsuturm L. Cyt b5 Prokaryotic expression Electron transfer

收稿日期: 2013-01-15 出版日期: 2013-04-25

ZTFLH:

Q819

基金资助: 中国科学院院地合作资助项目(XBXJ-2010-002)

通讯作者: 吴家和 E-mail: wujiahe@im.ac.cn

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引用本文:

刘金芝, 司怀军, 张宁, 吴家和. GhCyt b5基因的克隆及其蛋白参与电子传递功能的分析[J]. 中国生物工程杂志, 2013, 33(4): 68-73.

LIU Jin-zhi, SI Huai-jun, ZHANG Ning, WU Jia-he. Cloning and Expression Analysis of Cyt b5 Gene Involved in Electron Transfer in Gossypium hirsuturm. China Biotechnology, 2013, 33(4): 68-73.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/68

[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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