芥蓝1-脱氧-D-木酮糖-5-磷酸合成酶基因BaDXS1的克隆及原核表达

doi:10.3969/j.issn.1004-1524.2018.05.14

浙江农业学报 ›› 2018, Vol. 30 ›› Issue (5): 771-777.DOI: 10.3969/j.issn.1004-1524.2018.05.14

芥蓝1-脱氧-D-木酮糖-5-磷酸合成酶基因BaDXS1的克隆及原核表达

薛生玲¹, 江敏¹, 常嘉琪², 刘洋¹, 魏淋¹, 周建坤¹, 张芬¹, 孙勃^1,*

1.四川农业大学园艺学院,四川成都 611130;
2.浙江大学园艺系,浙江杭州 310058

收稿日期:2017-08-27 出版日期:2018-05-20 发布日期:2018-05-23
通讯作者: 孙勃,E-mail: 14099@sicau.edu.cn
作者简介:薛生玲(1993—),女,甘肃兰州人,硕士,研究方向为蔬菜生理与分子生物学。E-mail: 1181303872@qq.com
基金资助:
国家自然科学基金(31500247); 四川省省级大学生创新训练计划项目(201710626030)

Cloning and prokaryotic expression of 1-deoxy-D-xylulose-5-phosphate synthase encoding gene (BaDXS1) in Brassica alboglabra

XUE Shengling¹, JIANG Min¹, CHANG Jiaqi², LIU Yang¹, WEI Lin¹, ZHOU Jiankun¹, ZHANG Fen¹, SUN Bo^1,*

1. College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China;
2. Department of Horticulture, Zhejiang University, Hangzhou 310058, China

Received:2017-08-27 Online:2018-05-20 Published:2018-05-23

摘要/Abstract

摘要： 本研究以芥蓝为实验材料,采用同源克隆的方法分离出BaDXS1基因,其开放阅读框为2 139 bp,编码712个氨基酸。理化性质分析表明,BaDXS1蛋白的分子量为77.21 ku,等电点pI为8.59,不含跨膜区域,位于植物细胞叶绿体内。氨基酸序列比对发现,芥蓝DXS1与甘蓝、拟南芥、烟草、番茄等植物的DXS蛋白序列的一致性达到79%以上;系统进化树分析显示,芥蓝DXS1与甘蓝DXS的亲缘关系最近。构建原核表达载体pEASY-Blunt E1-BaDXS1,并对其进行诱导表达,发现该蛋白在大肠埃希菌体内主要以包涵体的形式存在。

关键词: 芥蓝, 1-脱氧-D-木酮糖-5-磷酸合成酶, 基因克隆, 原核表达

Abstract: The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) encoding gene BaDXS1 was isolated from Chinese kale (Brassica alboglabra) leaves by using homologous cloning method. BaDXS1 contained an open reading frame of 2 139 bp in length, encoding 712 amino acids. Physical and chemical properties analysis indicated that the molecular weight of BaDXS1 protein is 77.21 ku, and the isoelectric point pI is 8.59. There was no transmembrane region, and BaDXS1 is located in chloroplast of plant cell. Amino acid sequences comparison results showed that the identities of BaDXS1 and those in Brassica oleracea, Arabidopsis thaliana, tobacco, tomato were all over 79%. Phylogenetic tree analysis showed that it had close relation with BoDXS. The prokaryotic expression vector pEASY-Blunt E1-BaDXS1 was constructed, and the protein was successfully expressed in the form of inclusion bodies in Escherichia coli.

Key words: Chinese kale, 1-deoxy-D-xylulose-5-phosphate synthase, gene cloning, prokaryotic expression

中图分类号:

S637.2

薛生玲, 江敏, 常嘉琪, 刘洋, 魏淋, 周建坤, 张芬, 孙勃. 芥蓝1-脱氧-D-木酮糖-5-磷酸合成酶基因BaDXS1的克隆及原核表达[J]. 浙江农业学报, 2018, 30(5): 771-777.

XUE Shengling, JIANG Min, CHANG Jiaqi, LIU Yang, WEI Lin, ZHOU Jiankun, ZHANG Fen, SUN Bo. Cloning and prokaryotic expression of 1-deoxy-D-xylulose-5-phosphate synthase encoding gene (BaDXS1) in Brassica alboglabra[J]. , 2018, 30(5): 771-777.

参考文献

[1] 孙勃, 方莉, 刘娜, 等. 芥蓝不同器官主要营养成分分析[J]. 园艺学报, 2011, 38(3): 541-548.
SUN B, FANG L, LIU N, et al.Studies on main nutritional components of Chinese kale among different organs[J]. Acta Horticulturae Sinica, 2011, 38(3): 541-548. (in Chinese with English abstract)
[2] 孙勃, 张芬, 夏雪, 等. 芥蓝八氢番茄红素脱氢酶基因BaPDS1和BaPDS2的克隆与表达分析[J]. 园艺学报, 2016, 43(11): 2257-2265.
SUN B, ZHANG F, XIA X, et al.Cloning and expression analysis of BaPDS1 and BaPDS2 in Brassica alboglabra[J]. Acta Horticulturae Sinica, 2016, 43(11): 2257-2265. (in Chinese with English abstract)
[3] SMOLIKOVA G N, MEDVEDEV S S.Seed carotenoids: synthesis, diversity and functions[J]. Russian Journal of Plant Physiology, 2015, 62(1): 1-13.
[4] VRANOVÁ E, COMAN D, GRUISSEM W.Network analysis of the MVA and MEP pathways for isoprenoid synthesis[J]. Annual Review of Plant Biology, 2013, 64: 665-700.
[5] LOIS L M, RODRÍGUEZ-CONCEPCIÓN M, GALLEGO F, et al. Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-d-xylulose 5-phosphate synthase[J]. Plant Journal, 2000, 22(6): 503-513.
[6] ESTÉVEZ J M, CANTERO A, REINDL A, et al. 1-deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants[J]. Journal of Biological Chemistry, 2001, 276: 22901-22909.
[7] 潘夕春, 陈敏, 刘颜, 等. 番茄dxs基因的克隆及在大肠杆菌中的颜色互补[J]. 园艺学报, 2007, 34(2): 371-376.
PAN X C, CHEN M, LIU Y, et al.Cloning of deoxyoxylulose-5-phosphate synthase gene from tomato and its color complementation in E. coli[J]. Acta Horticulturae Sinica, 2007, 34(2): 371-376. (in Chinese with English abstract)
[8] CORDOBA E, PORTA H, ARROYO A, et al.Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize[J]. Journal of Experimental Botany, 2011, 62(6): 2023-2038.
[9] 魏麟, 伍贤进, 李胜华, 等. 鱼腥草1-脱氧-D-木酮糖-5-磷酸合成酶1基因克隆与表达分析[J]. 中草药, 2014, 45(11): 1607-1612.
WEI L, WU X J, LI S H, et al.Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthase 1 gene in Houttuynia cordata[J]. Chinese Traditional and Herbal Drugs, 2014, 45(11): 1607-1612. (in Chinese with English abstract)
[10] 孙君, 陈桂信, 叶乃兴, 等. 茉莉花香气相关基因JsDXS基因及其启动子的克隆与表达分析[J]. 园艺学报, 2014, 41(6): 1236-1244.
SUN J, CHEN G X, YE N X, et al.Cloning and expression analysis of deoxyoxylulose-5-phosphate synthase gene related to aroma from Jasminum sambac and isolation of its promoter[J]. Acta Horticulturae Sinica, 2014, 41(6): 1236-1244. (in Chinese with English abstract)
[11] 王毅, 周旭, 毕玮, 等. 思茅松1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析[J]. 林业科学研究, 2015, 28(6): 833-838.
WANG Y, ZHOU X, BI W, et al.Identification and characterization of a 1-deoxy-D-xylulose-5-phosphate synthase gene from Pinus kesiya var. langbianensis[J].Forest Research, 2015, 28(6): 833-838. (in Chinese with English abstract)
[12] HENRIQUEZ M A, SOLIMAN A, LI G, et al.Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans[J]. Plant Science, 2016, 243: 71-83.
[13] CHEN Q, YU H W, WANG X R, et al.An alternative cetyltrimethylammonium bromide-based protocol for RNA isolation from blackberry (Rubus L.)[J]. Genetics and Molecular Research, 2011, 11(2): 1773-1782.
[14] 黄路平, 刘仑, 鲁黎明, 等. 烟草NtKAT3基因克隆、序列和表达分析[J]. 浙江农业学报, 2017, 29(7): 1057-1063.
HUANG L P, LIU L, LU L M, et al.Cloning, sequence and expression analysis of a potassium channel NtKAT3 in tobacco[J]. Acta Agriculturae Zhejiangensis, 2017, 29(7): 1057-1063. (in Chinese with English abstract)
[15] 任学良, 李立芹, 许力, 等. 烟草糖基转移酶基因NtGT3的克隆与生物信息学分析[J]. 浙江农业学报, 2017, 29(1): 23-30.
REN X L, LI L Q, XU L, et al.Cloning and bioinformatic analysis of glucosetransferase gene NtGT3 in Nicotiana tabacum[J]. Acta Agriculturae Zhejiangensis, 2017, 29(1): 23-30. (in Chinese with English abstract)
[16] LANGE B M, WILDUNG M R, MCCASKILL D, et al.A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(5): 2100-2104.
[17] LOIS L M, CAMPOS N, PUTRA S R, et al.Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(5): 2105-2110.
[18] 尹春光, 杜立新, 赵桂平, 等. Mx基因稀有密码子和mRNA结构及大肠杆菌表达优化[J]. 遗传, 2009, 31(1): 75-82.
YIN C G, DU L X, ZHAO G P, et al.Optimizing the expression of Mx gene in Escherichia coil based on rare codon and mRNA structure[J]. Hereditas, 2009, 31(1): 75-82. (in Chinese with English abstract)
[19] 薛生玲, 张芬, 江敏, 等. 芥蓝八氢番茄红素合成酶基因BaPSY1的克隆及原核表达[J]. 基因组学与应用生物学, 2017, 36(2): 734-739.
XUE S L, ZHANG F, JIANG M, et al.Cloning and prokaryotic expression of phytoene synthase gene in Brassica alboglabra[J]. Genomics and Applied Biology, 2017, 36(2): 734-739. (in Chinese with English abstract)
[20] 刘永强, 王熙然, 叶云天, 等. 草莓FaUVR8基因的cDNA克隆、生物信息学分析及原核表达条件优化[J]. 分子植物育种, 2016, 14(11): 2964-2975.
LIU Y Q, WANG X R, YE Y T, et al.Cloning, bioinformatic analysis of strawberry FaUVR8 gene and optimization of its prokaryotic expression conditions[J]. Molecular Plant Breeding, 2016, 14(11): 2964-2975. (in Chinese with English abstract)
[21] RODRÍGUEZ-CONCEPCIÓN M, BORONAT A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics[J]. Plant Physiology, 2002, 130(3): 1079-1089.
[22] PENG G, WANG C, SONG S, et al.The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation[J]. Plant Physiology & Biochemistry, 2013, 71(2): 67-76.

芥蓝1-脱氧-D-木酮糖-5-磷酸合成酶基因BaDXS1的克隆及原核表达

Cloning and prokaryotic expression of 1-deoxy-D-xylulose-5-phosphate synthase encoding gene (BaDXS1) in Brassica alboglabra

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