甜菜M14品系BvM14-UNG基因克隆及生物信息学分析

doi:10.11924/j.issn.1000-6850.casb2021-0889

中国农学通报 ›› 2022, Vol. 38 ›› Issue (4): 16-22.doi: 10.11924/j.issn.1000-6850.casb2021-0889

所属专题：生物技术；油料作物；园艺

甜菜M14品系BvM14-UNG基因克隆及生物信息学分析

王盛昊¹^,²(), 于冰¹^,²()

¹黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 150500
²黑龙江大学生命科学学院/黑龙江省普通高校分子生物学重点实验室,哈尔滨 150080

收稿日期:2021-09-16 修回日期:2021-11-23 出版日期:2022-02-05 发布日期:2022-03-16
通讯作者: 于冰
作者简介:王盛昊,男,1997年出生,山东聊城人,硕士研究生,研究方向：植物分子生物学。通信地址：150080 黑龙江省哈尔滨市学府路74号黑龙江大学生命科学学院320室,E-mail： Wangshenghao0528@163.com。
基金资助:
中国博士后项目“甜菜M14品系BvM14-STPK互作蛋白鉴定及其耐盐功能研究”(204968)

Cloning and Bioinformatics Analysis of BvM14-UNG Gene in Sugarbeet M14 Line

WANG Shenghao¹^,²(), YU Bing¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
²Key Laboratory of Molecular Biology, College of Heilongjiang Province/ School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2021-09-16 Revised:2021-11-23 Online:2022-02-05 Published:2022-03-16
Contact: YU Bing

摘要/Abstract

摘要：

为研究Bv-UNG蛋白在植物碱基切除修复(base-excision repair,BER)途径中的功能,以甜菜M14品系为材料,根据NCBI上二倍体栽培甜菜UDG编码基因Bv-UNG序列,利用同源序列克隆法获得甜菜M14品系BvM14-UNG基因cDNA全长,并对其进行生物信息学分析。结果表明,该基因编码374个氨基酸,BvM14-UNG蛋白表现出较强的亲水性,为可溶性蛋白,主要由无规卷曲和α螺旋构成,具有水激活基序、尿嘧啶结合基序、小沟插入环基序、尿嘧啶糖基化酶抑制剂(uracil glycosylase inhibitor,UGI)结合位点和亮氨酸(Leu)位点,属于UDG-F1亚家族,BvM14-UNG蛋白与甜菜BvUNG蛋白亲缘性最近,其次为藜麦CqUNG蛋白。本研究可为后续开展甜菜M14品系BER途径中BvM14-UNG酶活性研究提供参考。

关键词: 甜菜M14品系, BvM14-UNG基因, 碱基切除修复, 基因克隆, 生物信息学分析

Abstract:

To research the function of Bv-UNG protein in base excision repair (BER) pathway, the full-length cDNA of BvM14-UNG gene of sugarbeet M14 line was obtained by homologous sequence cloning method according to the sequence of UDG coding gene Bv-UNG of diploid cultivated sugarbeet on NCBI and its bioinformatics analysis was carried out. The results show that the gene encodes 374 amino acids, the BvM14-UNG protein shows strong hydrophilicity and is a soluble protein, which is mainly composed of random coils and alpha helices, and has water-activated motif, uracil binding motif, minor groove insertion loop motif, uracil glycosylase inhibitor (UGI) binding site and leucine (Leu) site. BvM14-UNG belongs to UDG-F1 family. BvM14-UNG protein is closely related to BvUNG protein, followed by CqUNG protein. This study lays a foundation for the follow-up study on BvM14-UNG enzyme activity of sugar beet M14 line in BER pathway.

Key words: sugarbeet M14 line, BvM14-UNG gene, base-excision repair (BER), gene clone, bioinformatics analysis

中图分类号:

S566.3

王盛昊, 于冰. 甜菜M14品系BvM14-UNG基因克隆及生物信息学分析[J]. 中国农学通报, 2022, 38(4): 16-22.

WANG Shenghao, YU Bing. Cloning and Bioinformatics Analysis of BvM14-UNG Gene in Sugarbeet M14 Line[J]. Chinese Agricultural Science Bulletin, 2022, 38(4): 16-22.

图/表 10

引物名称	序列
BvM14-UNG-S	5'-CGTCAAAGAAATGAAATCTCTCCGA-3'
BvM14-UNG-AS	5'-GCTTCAGGCACAAGTCATTCCAT-3'

表1. 实验所需引物

图1. 盐胁迫下甜菜M14品系根总RNA的1%琼脂糖凝胶电泳图

图2. BvM14-UNG基因cDNA全长PCR扩增产物的1%琼脂糖凝胶电泳图 M为DL2000 Marker,1~3为BvM14-UNG基因cDNA全长PCR扩增产物

图3. BvM14-UNG基因cDNA全长开放阅读框分析

图4. BvM14-UNG蛋白亲/疏水性预测结果

图5. BvM14-UNG蛋白的二级结构预测结果

图6. BvM14-UNG蛋白的三级结构预测结果红色代表水激活基序(MotifⅠ),绿色代表尿嘧啶结合基序(MotifⅡ),棕色代表小沟插入环基序(MotifⅢ),紫色代表UGI结合位点,黑色代表Leu位点

图7. BvM14-UNG保守结构域分析

图8. BvM14-UNG蛋白多重序列比对结果

图9. BvM14-UNG蛋白系统进化树分析

参考文献 26

[1]	KROKAN H E, DRABLØS F, SLUPPHAUG G. Uracil in DNA-occurrence, consequences and repair[J]. Oncogene, 2002, 21(58):8935-48. doi: 10.1038/sj.onc.1205996 URL
[2]	GUILLET M, BOITEUX S. Origin of endogenous DNA abasic sites in saccharomyces cerevisiae[J]. Molecular and cellular biology, 2003, 23(22):8386-94. doi: 10.1128/MCB.23.22.8386-8394.2003 URL
[3]	CÓRDOBA-CAÑERO D, DUBOIS E, ARIZA R R, et al. Arabidopsis uracil DNA glycosylase (UNG) is required for base excision repair of uracil and increases plant sensitivity to 5-fluorouracil[J]. The Journal of biological chemistry, 2010, 285(10):7475-83. doi: 10.1074/jbc.M109.067173 URL
[4]	GUILLET M, VAN DER KEMP P A, Boiteux S. dUTPase activity is critical to maintain genetic stability in Saccharomyces cerevisiae[J]. Nucleic acids research, 2006, 34(7):2056-66. doi: 10.1093/nar/gkl139 URL
[5]	YI G S, WANG W W, CAO W G, et al. Sulfolobus acidocaldarius UDG can Remove dU from the RNA Backbone: Insight into the specific recognition of Uracil linked with deoxyribose[J]. Genes, 2017, 8(1).
[6]	FORTINI P, DOGLIOTTI E. Base damage and single-strand break repair: mechanisms and functional significance of short- and long-patch repair subpathways[J]. DNA repair, 2007, 6(4):398-409. doi: 10.1016/j.dnarep.2006.10.008 URL
[7]	LINDAHL T. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues[J]. Proceedings of the national academy of sciences of the United States of America, 1974, 71(9):3649-3653.
[8]	鲍洪波, 王晋芳, 钱世钧. 尿嘧啶N糖基化酶(UNG)的研究进展[J]. 中国生物工程杂志, 2003(1):43-7.
[9]	YOUSIF A S, STANLIE A, BEGUM N A. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination[J]. International immunology, 2014, 26(10):575-578. doi: 10.1093/intimm/dxu071 URL
[10]	KROKAN H E, OTTERLEI M, NILSEN H, et al. Properties and functions of human uracil-DNA glycosylase from the UNG gene[J]. Progress in nucleic acid research and molecular biology, 2001, 68:365-386.
[11]	KARWOWSKI B T. The influence of (5'R)- and (5'S)-5',8-cyclo-2'-deoxyadenosine on UDG and hAPE1 activity. Tandem lesions are the base excision repair system's nightmare[J]. Cells, 2019, 8(11).366-377 doi: 10.3390/cells8040366 URL
[12]	TRAVERS A, MUSKHELISHVILI G. DNA structure and function[J]. Bioessays news & reviews in molecular cellular & developmental biology, 2015, 282(12):2279-2295.
[13]	PEARL L H. Structure and function in the uracil-DNA glycosylase superfamily[J]. Mutation research, 2000, 460(3-4):165-181. doi: 10.1016/S0921-8777(00)00025-2 URL
[14]	KROKAN H E, OTTERLEI M, NILSEN H, et al. Properties and functions of human uracil-DNA glycosylase from the UNG gene[J]. Progress in nucleic acid research and molecular biology, 2001, 68:365-386.
[15]	FORTINI P, FERRETTI C, DOGLIOTTI E. The response to DNA damage during differentiation: pathways and consequences[J]. Mutation research, 2013, 743-744:160-168. doi: 10.1016/j.mrfmmm.2013.03.004 URL
[16]	BLAISDELL P, WARNER H. Partial purification and characterization of a uracil-DNA glycosylase from wheat germ[J]. The journal of biological chemistry, 1983, 258(3):1603-1609. doi: 10.1016/S0021-9258(18)33027-8 URL
[17]	BONES A M. Expression and occurrence of uracil-DNA glycosylase in higher plants[J]. Physiologia plantarum, 1993, 88(4):682-688. doi: 10.1111/ppl.1993.88.issue-4 URL
[18]	郭德栋, 方晓华, 刘丽萍, 等. 无融合生殖甜菜单体附加系的获得和鉴定[J]. 云南大学学报:自然科学版, 1999(S3):180-181.
[19]	戈岩, 何光存, 王志伟, 等. 无融合生殖甜菜M14的GISH和BAC-FISH研究[J]. 中国科学, 2007(2):209-216.
[20]	LI J, LI H, YANG N, et al. Overexpression of a monodehydroascorbate reductase gene from sugar beet M14 increased salt stress tolerance[J]. Sugar tech, 2021, 23(1):45-56. doi: 10.1007/s12355-020-00877-0 URL
[21]	WANG Y, ZHAN Y, WU C, et al. Cloning of a cystatin gene from sugar beet M14 that can enhance plant salt tolerance[J]. Plant science, 2012, 191-192:93-99. doi: 10.1016/j.plantsci.2012.05.001 URL
[22]	WU C, MA C, PAN Y, et al. Sugar beet M14 glyoxalase I gene can enhance plant tolerance to abiotic stresses[J]. Journal of plant research, 2013, 126(3):415-425. doi: 10.1007/s10265-012-0532-4 URL
[23]	赵冬美, 张咏雪, 李海英. 甜菜M14品系BvM14-STPK基因的生物信息学分析及响应盐胁迫的表达分析[J]. 中国农学通报, 2020, 36(1):35-42.
[24]	CHATTERJEE N, WALKER G C. Mechanisms of DNA damage, repair, and mutagenesis[J]. Environmental and molecular mutagenesis, 2017, 58(5):235-263. doi: 10.1002/em.v58.5 URL
[25]	HUFFMAN J L, SUNDHEIM O, TAINER J A. DNA base damage recognition and removal: new twists and grooves[J]. Mutation research, 2005, 577(1-2):55-76. doi: 10.1016/j.mrfmmm.2005.03.012 URL
[26]	BACOLLA A, SENGUPTA S, YE Z, et al. Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin[J]. Nucleic acids research, 2021, 49(1):221-243. doi: 10.1093/nar/gkaa1120 URL

甜菜M14品系BvM14-UNG基因克隆及生物信息学分析

Cloning and Bioinformatics Analysis of BvM14-UNG Gene in Sugarbeet M14 Line

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