棉花GhAO3基因的克隆、功能序列分析及原核表达

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 51-56.

棉花GhAO3基因的克隆、功能序列分析及原核表达

冉茂飞¹, 贺怡¹, 钱雯婕¹, 王斐¹, 李鸿彬^1，2

（1. 石河子大学生命科学学院，石河子 832000；2. 石河子大学农业技术重点实验室，石河子 832000）

收稿日期:2013-02-26 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:冉茂飞，男，硕士研究生，研究方向：植物分子生物学；E-mail：1069585529@qq.com
基金资助:
国家自然科学基金项目（31260039），农业部转基因专项（2009ZX08005-027B），兵团种质资源创新专项（2012BB050）

Cloning，Functional Sequence Analysis and Prokaryotic Expression of Cotton GhAO3 cDNA

Ran Maofei¹, He Yi¹, Qian Wenjie¹, Wang Fei¹, Li Hongbin^{1, 2}

（1. College of Life Science of Shihezi University，Shihezi 832000；2. Key Laboratory of Agrobiotechnology of Shihezi University，Shihezi 832000）

Received:2013-02-26 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要： 通过RT-PCR方法从处于快速伸长发育时期（开花后15 d）的棉花纤维组织中克隆得到棉花抗坏血酸氧化酶3(Ascorbate oxidase 3, GhAO3)基因的全长开放读码框，该基因全长读码框为1 758 bp，编码包含585个氨基酸的蛋白质。通过序列功能分析和同源序列比对，GhAO3蛋白具有较高的保守性，包括3个多铜氧化酶结构域以及跨膜信号序列，进化树比对结果显示GhAO3与大豆GmAO的亲缘关系较近。将GhAO3基因与原核表达载体pET32a相连构建重组载体pET32a-GhAO3，把重组载体转入大肠杆菌BL21（DE3）中，用IPTG诱导重组菌，经SDS-PAGE电泳分析，获得分子量约为64 kD的重组GhAO3蛋白。

关键词: 棉花抗坏血酸氧化酶, 基因克隆, 功能序列分析, 原核表达

Abstract: A cotton ascorbate oxidase(GhAO3)full-length open reading frame（ORF）cDNA was cloned from elongating fiber tissue（15 DPA）by RT-PCR method, GhAO3 ORF contains 1 758 bp nucleotides and codes a protein of 585 amino acids. Through sequence function analysis and homology sequence alignment, GhAO3 protein includes three multi-copper oxidase domain and transmembrane signal sequence with a high conservation. Phylogenetic tree analysis showed that GhAO3 has a similar relationship with GmAO protein. Prokaryotic expression vector pET32a-GhAO3 was constructed and transformated into E. coli BL21（DE3）. Recombinant GhAO3 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD, and had a higher enzymatic activity.

Key words: Cotton ascorbate oxidase, Gene cloning, Functional sequence analysis, Prokaryotic expression

冉茂飞, 贺怡, 钱雯婕, 王斐, 李鸿彬. 棉花GhAO3基因的克隆、功能序列分析及原核表达[J]. 生物技术通报, 2013, 0(8): 51-56.

Ran Maofei, He Yi, Qian Wenjie, Wang Fei, Li Hongbin. Cloning，Functional Sequence Analysis and Prokaryotic Expression of Cotton GhAO3 cDNA[J]. Biotechnology Bulletin, 2013, 0(8): 51-56.

参考文献

[1] Tullio MC. Ascorbic acid oxidase：an enzyme in search of a role[J]. Biologia Plantarum, 2004, 48（2）：161-166.
[2] Sanmartin M, Drogoudi PD, Lyons T. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone[J]. Planta, 2003, 216：918-928.
[3] Sanmartin M, Pateraki I. Diverential expression of the ascorbate oxidase multigene family during fruit development and in response to stress[J]. Planta, 2007, 225：873-885.
[4] Kato N, Esaka M. Expression of transgenic tobacco protoplasts expressiong Pumpkin asocbrate oxidase is more rapid than that of wild-tpye protoplasts[J]. Plant, 2000, 210：1018-1022.
[5] Kato N, Esaka M. Changes in asocrbate oxidase gene expression and ascorbatelevels in cell division and cell elongation in tobaocc cells[J]. Physiol Plant, 1999, 105：321-329.
[6] Li HB, Qin YM, Pang Y, et al. A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell development[J]. New Phytologist, 2007, 175：462-471.
[7] 蒋建雄, 张天真.利用CTAB/酸酚法提取棉花组织总RNA[J].棉花学报, 2003, 15：166-167.
[8] 胡根海, 喻树迅.利用改良的CTAB法提取棉花叶片总RNA[J].棉花学报, 2007, 19（1）：69-70.
[9] 陈坤明, 宫海军, 王锁民.植物抗坏血酸的生物合成、转运及生物学功能[J].西北植物学报, 2004, 24（2）：329-336.
[10] Conklin PL, Saracco SA, Norris SR, Last RL. Identification of ascorbic acid-deficient Arabidopsis thaliana mutants[J]. Genetics, 2000, 154：847-856.
[11] Sanmartin M, Drogoudi PA, Lyons T, et al. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone[J]. Planta, 2003, 216（6）：918-928.
[12] Glen L, Mark A, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants[J]. Nature, 1998, 393：365-369.
[13] Ohkawa J, Okada N, Shinmyo A, Takano M. Primary structure of cucumbei(Cucumis sativus)ascorbate oxidase deduced from cDNA sequences bomology with blue copper proteins and tissue-specific expression[J]. Proc Natl Acad Sci USA, 1989, 86（4）：1239-1243.
[14] Mol J. Refined crystal structure of ascorbate oxidase at 1.9 A resolution[J]. Biol, 1992, 224（1）：179-205.
[15] Sanmartin M, Pavlina D, Lyons T. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone[J]. Planta, 2003, 216：918-928.
[16] Yammaoto A, Bhuiyan NH, Waditee R, et al. Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants[J]. J Exp Bot, 2005, 56：1785-1796.
[17] Pignocchi C, John M. The function of ascorbate oxidase in tobacco[J]. Plant Physiology, 2003, 132：1631-1641.