甜瓜BZR转录因子家族基因的全基因组鉴定及表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0162

摘要/Abstract

摘要：

【目的】 油菜素唑抗性因子（brassinazole-resistant，BZR）是植物特有的转录因子，对植物的生长发育起着至关重要的作用。从甜瓜全基因组范围内鉴定CmBZR基因家族成员，分析其相关基因的表达模式，为进一步探究甜瓜BZR基因家族的生物学功能提供基础。【方法】 基于甜瓜全基因组数据，通过BLAST对甜瓜BZR家族基因进行鉴定，利用生物信息学的方法分析了该家族蛋白的理化性质、基因染色体分布、基因结构、蛋白质结构域、系统进化以及启动子顺式作用元件，并利用荧光定量PCR验证BZR家族基因在甜瓜不同组织及不同生长发育时期果实中的表达情况。【结果】 甜瓜中共鉴定到6个BZR基因，系统进化分析可将其分为5个亚家族，CmBZR基因分布于第3、7、8、11和12染色体上。所有的BZR蛋白质都具有保守的结构域。CmBZR基因启动子区域显著富集与生长发育、激素信号转导和非生物逆境胁迫相关的顺式作用元件。CmBEH1-4在茎、两性花以及子房中高表达，在生长期、成熟期、呼吸跃变期和呼吸跃变后期果实中也均有表达。【结论】 在全基因组范围内从甜瓜中系统鉴定出6个甜瓜CmBZR基因家族成员，不同基因在甜瓜的各个组织和不同的生长发育时期均有表达，且表达模式存在差异。

关键词: 甜瓜, BZR, 转录因子, 全基因组鉴定, 表达分析, 生物信息学

Abstract:

【Objective】 Brassinazole-resistant（BZR）is a plant specific transcription factor that plays a crucial role in plant growth and development. Identifying members of the CmBZR gene family across the whole melon genome, and analyzing the expression patterns of related gene, may provide a basis for further exploring the biological functions of the melon BZR gene family. 【Method】 Based on the whole melon genome data, the BZR family genes in melon were identified using BLAST. Bioinformatics methods were used to analyze the physicochemical properties, chromosomal distribution, gene structure, protein domains, phylogenetic evolution, and cis acting elements of the family proteins. Fluorescence quantitative PCR was used to verify the expression of BZR family genes in different tissues and fruits of melon at different growth and development stages. 【Result】 A total of 6 BZR genes were identified in cantaloupe, which can be divided into 5 subfamilies through phylogenetic analysis. CmBZR genes are distributed on chromosomes 3, 7, 8, 11, and 12. All BZR proteins have conserved domains. The promoter region of the CmBZR gene is significantly enriched with cis-acting elements related to growth and development, hormone signal transduction, and abiotic stress. CmBEH1-4 is highly expressed in stems, female flowers, and ovaries, and is also expressed in growth, maturity, respiratory climacteric, and late respiratory climacteric stages. 【Conclusion】 Six members of the melon CmBZR gene family have been systematically identified across the whole genome, and different genes are expressed in various tissues and growth stages of the melon，and their expression patterns are various.

Key words: melon（Cucumis melo L.）, BZR, transcription factor, genome-wide identification, expression analysis, bioinformatics

臧文蕊, 马明, 砗根, 哈斯阿古拉. 甜瓜BZR转录因子家族基因的全基因组鉴定及表达模式分析[J]. 生物技术通报, 2024, 40(7): 163-171.

ZANG Wen-rui, MA Ming, CHE Gen, HASI Agula. Genome-wide Identification and Expression Pattern Analysis of BZR Transcription Factor Gene Family of Melon[J]. Biotechnology Bulletin, 2024, 40(7): 163-171.

图/表 6

表1 甜瓜BZR家族基本信息

Table 1 Basic information of BZR family members in melon

基因名称 Gene name	登录号Accession No.	染色体分布Chromosome distribution	染色体座位Chromosomal loci/bp	ORF /bp	氨基酸数量Number of amino acids	分子量Molecular weight/kD	等电点Isoelectric point（pI）
CmBZR1	MELO3C010925.2.1	Chr 03	29999092-30000475	936	311	34.05	9.16
CmBES1	MELO3C007804.2.1	Chr 08	5468519-5471484	960	319	34.71	8.96
CmBEH1	MELO3C016213.2.1	Chr 07	22268773-22275513	1 932	643	75.11	5.90
CmBEH2	MELO3C021214.2.1	Chr 11	31280641-31285843	2 046	681	78.21	5.74
CmBEH3	MELO3C016121.2.1	Chr 07	20822745-20826732	978	325	34.70	8.50
CmBEH4	MELO3C002681.2.1	Chr 12	22058946-22061841	984	327	39.52	9.00

图1 CmBZR蛋白序列分析 A：6个CmBZR蛋白的多序列比对；B：CmBZR序列中BZR结构域的保守位点。*：CmBZR蛋白序列保守基序

Fig. 1 Analysis of CmBZR protein sequences A: Multiple sequence alignment of 6 CmBZR proteins; B: conserved sites of BZR domains in CmBZR sequences. *: Conserved motif of CmBZR protein sequences

图2 甜瓜BZR基因结构分析

Fig. 2 Gene structure analysis of BZR genes in melon

图3 拟南芥、水稻、番茄、黄瓜、南瓜和甜瓜中BZR蛋白的系统发育树分析 I-V为6个物种BZR家族成员的5个亚家族

Fig. 3 Phylogenetic tree analysis of BZR proteins from Arabidopsis，Oryza sativa，Solanum lycopersicon，Cucumis sativus，Cucurbita moschata and C. smelo I-V indicates different subfamily of BZR members in six species

图4 CmBZR基因家族启动子区域顺式作用元件

Fig. 4 Cis-elements of promoter region of CmBZR gene family in melon

图5 CmBZR基因的表达模式分析 A：6个CmBZR基因在甜瓜不同组织中表达的热图；B：CmBZR基因在甜瓜Rt、S、L、FF、MF、O、G、R、C和P中的表达谱，这些字母分别代表根、茎、叶、两性花、雄花、子房、生长期、成熟期、呼吸跃变期呼吸跃变后期果实

Fig. 5 Expression analysis of CmBZR in melon A: Heat map of 6 CmBZR genes expressed differently in different tissues of melon；B: expression profiles of CmBZR genes in melon’s Rt, S, L, FF, MF, O, G,R, C and P, and they indicate the root, stem, leaf, female flower, male flower, ovary, growing stage, ripening stage, climacteric stage, and post-climacteric stage fruit, respectively.

参考文献 45

[1]	Shin AY, Kim YM, Koo N, et al. Transcriptome analysis of the oriental melon(Cucumis melo L. var. makuwa)during fruit development[J]. PeerJ, 2017, 5: e2834.
[2]	Yin YH, Vafeados D, Tao Y, et al. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis[J]. Cell, 2005, 120(2): 249-259.
[3]	Steber CM, McCourt P. A role for brassinosteroids in germination in Arabidopsis[J]. Plant Physiol, 2001, 125(2): 763-769. doi: 10.1104/pp.125.2.763 pmid: 11161033
[4]	Yang CJ, Zhang C, Lu YN, et al. The mechanisms of brassinosteroids'action: from signal transduction to plant development[J]. Mol Plant, 2011, 4(4): 588-600.
[5]	Galstyan A, Nemhauser JL. Auxin promotion of seedling growth via ARF5 is dependent on the brassinosteroid-regulated transcription factors BES1 and BEH4[J]. Plant Direct, 2019, 3(9): e00166.
[6]	Liang T, Mei SL, Shi C, et al. UVR8 interacts with BES1 and BIM1 to regulate transcription and photomorphogenesis in Arabidopsis[J]. Dev Cell, 2018, 44(4): 512-523.e5. doi: S1534-5807(17)31079-1 pmid: 29398622
[7]	Wang WX, Lu XD, Li L, et al. Photoexcited CRYPTOCHROME1 interacts with dephosphorylated BES1 to regulate brassinosteroid signaling and photomorphogenesis in Arabidopsis[J]. Plant Cell, 2018, 30(9): 1989-2005.
[8]	Bajguz A, Hayat S. Effects of brassinosteroids on the plant responses to environmental stresses[J]. Plant Physiol Biochem, 2009, 47(1): 1-8.
[9]	Liao K, Peng YJ, Yuan LB, et al. Brassinosteroids antagonize jasmonate-activated plant defense responses through BRI1-EMS-SUPPRESSOR1(BES1)[J]. Plant Physiol, 2020, 182(2): 1066-1082. doi: 10.1104/pp.19.01220 pmid: 31776183
[10]	Cui XY, Gao Y, Guo J, et al. BES/BZR transcription factor TaBZR2 positively regulates drought responses by activation of TaGST1[J]. Plant Physiol, 2019, 180(1): 605-620.
[11]	Jaillais Y, Hothorn M, Belkhadir Y, et al. Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor[J]. Genes Dev, 2011, 25(3): 232-237.
[12]	Tang WQ, Kim TW, Oses-Prieto JA, et al. BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis[J]. Science, 2008, 321(5888): 557-560.
[13]	Kim TW, Guan SH, Burlingame AL, et al. The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2[J]. Mol Cell, 2011, 43(4): 561-571.
[14]	Peng P, Yan ZY, Zhu YY, et al. Regulation of the Arabidopsis GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 through proteasome-mediated protein degradation[J]. Mol Plant, 2008, 1(2): 338-346. doi: 10.1093/mp/ssn001 pmid: 18726001
[15]	Tang WQ, Yuan M, Wang RJ, et al. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1[J]. Nat Cell Biol, 2011, 13(2): 124-131.
[16]	Yin YH, Wang ZY, Mora-Garcia S, et al. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation[J]. Cell, 2002, 109(2): 181-191. doi: 10.1016/s0092-8674(02)00721-3 pmid: 12007405
[17]	Wang ZY, Nakano T, Gendron J, et al. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis[J]. Dev Cell, 2002, 2(4): 505-513. doi: 10.1016/s1534-5807(02)00153-3 pmid: 11970900
[18]	Bai MY, Zhang LY, Gampala SS, et al. Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice[J]. Proc Natl Acad Sci USA, 2007, 104(34): 13839-13844.
[19]	Gampala SS, Kim TW, He JX, et al. An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis[J]. Dev Cell, 2007, 13(2): 177-189.
[20]	Szekeres M, Németh K, Koncz-Kálmán Z, et al. Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis[J]. Cell, 1996, 85(2): 171-182. doi: 10.1016/s0092-8674(00)81094-6 pmid: 8612270
[21]	Gil P, Liu Y, Orbović V, et al. Characterization of the auxin-inducible SAUR-AC1 gene for use as a molecular genetic tool in Arabidopsis[J]. Plant Physiol, 1994, 104(2): 777-784. doi: 10.1104/pp.104.2.777 pmid: 8159792
[22]	Xie LQ, Yang CJ, Wang XL. Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis[J]. J Exp Bot, 2011, 62(13): 4495-4506.
[23]	Ibañez C, Delker C, Martinez C, et al. Brassinosteroids dominate hormonal regulation of plant thermomorphogenesis via BZR1[J]. Curr Biol, 2018, 28(2): 303-310.e3. doi: S0960-9822(17)31602-0 pmid: 29337075
[24]	Oh E, Zhu JY, Wang ZY. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses[J]. Nat Cell Biol, 2012, 14(8): 802-809. doi: 10.1038/ncb2545 pmid: 22820378
[25]	Chen LG, Gao ZH, Zhao ZY, et al. BZR1 family transcription factors function redundantly and indispensably in BR signaling but exhibit BRI1-independent function in regulating anther development in Arabidopsis[J]. Mol Plant, 2019, 12(10): 1408-1415.
[26]	于好强, 孙福艾, 冯文奇, 等. 转录因子BES1/BZR1调控植物生长发育及抗逆性[J]. 遗传, 2019, 41(3): 206-214.
	Yu HQ, Sun FA, Feng WQ, et al. The BES1/BZR1 transcription factors regulate growth, development and stress resistance in plants[J]. Hereditas, 2019, 41(3): 206-214.
[27]	He JX, Gendron JM, Sun Y, et al. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses[J]. Science, 2005, 307(5715): 1634-1638.
[28]	Luo SL, Zhang GB, Zhang ZY, et al. Genome-wide identification and expression analysis of BZR gene family and associated responses to abiotic stresses in cucumber(Cucumis sativus L.)[J]. BMC Plant Biol, 2023, 23(1): 214.
[29]	李春, 刘小俊, 蔡鹏, 等. 中国南瓜BZR基因家族的全基因组鉴定及生物信息学分析[J]. 分子植物育种, 2022, 20(19): 6324-6330.
	Li C, Liu XJ, Cai P, et al. Genome-wide identification and bioinformatics analysis of BZR gene family in pumpkin(Cucurbita moschata duch.)[J]. Mol Plant Breed, 2022, 20(19): 6324-6330.
[30]	陈旭, 沈春洋, 莫福磊, 等. 番茄BZR基因家族鉴定及非生物胁迫下表达模式分析[J]. 东北农业大学学报, 2021, 52(11): 9-17.
	Chen X, Shen CY, Mo FL, et al. Identification of BZR gene family in tomato and expression patterns analysis under abiotic stress[J]. J Northeast Agric Univ, 2021, 52(11): 9-17.
[31]	Zheng Y, Wu S, Bai Y, et al. Cucurbit Genomics Database(CuGenDB): a central portal for comparative and functional genomics of cucurbit crops[J]. Nucleic Acids Res, 2019, 47(D1): D1128-D1136.
[32]	Johnson LS, Eddy SR, Portugaly E. Hidden Markov model speed heuristic and iterative HMM search procedure[J]. BMC Bioinformatics, 2010, 11: 431. doi: 10.1186/1471-2105-11-431 pmid: 20718988
[33]	Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020[J]. Nucleic Acids Res, 2021, 49(D1): D458-D460.
[34]	Wilkins MR, Gasteiger E, Bairoch A, et al. Protein identification and analysis tools in the ExPASy server[J]. Methods Mol Biol, 1999, 112: 531-552. pmid: 10027275
[35]	Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11[J]. Mol Biol Evol, 2021, 38(7): 3022-3027. doi: 10.1093/molbev/msab120 pmid: 33892491
[36]	Waterhouse AM, Procter JB, Martin DMA, et al. Jalview Version 2—a multiple sequence alignment editor and analysis workbench[J]. Bioinformatics, 2009, 25(9): 1189-1191. doi: 10.1093/bioinformatics/btp033 pmid: 19151095
[37]	Crooks GE, Hon G, Chandonia JM, et al. WebLogo: a sequence logo generator[J]. Genome Res, 2004, 14(6): 1188-1190. doi: 10.1101/gr.849004 pmid: 15173120
[38]	Bolser D, Staines DM, Pritchard E, et al. Ensembl plants: integrating tools for visualizing, mining, and analyzing plant genomics data[J]. Methods Mol Biol, 2016, 1374: 115-140. doi: 10.1007/978-1-4939-3167-5_6 pmid: 26519403
[39]	Chen CJ, Chen H, Zhang Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202. doi: S1674-2052(20)30187-8 pmid: 32585190
[40]	江倩倩, 王雨婷, 惠竹梅. 葡萄BZR基因家族的鉴定及表达分析[J]. 植物生理学报, 2021, 57(6): 1218-1228.
	Jiang QQ, Wang YT, Hui ZM. Identification and expression analysis of BZR gene family in grapevine[J]. Plant Physiol J, 2021, 57(6): 1218-1228.
[41]	Manoli A, Trevisan S, Quaggiotti S, et al. Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L[J]. Plant Growth Regul, 2018, 84(3): 423-436.
[42]	Sarwar R, Geng R, Li L, et al. Genome-wide prediction, functional divergence, and characterization of stress-responsive BZR transcription factors in B. napus[J]. Front Plant Sci, 2022, 12: 790655.
[43]	Li YY, He LL, Li J, et al. Genome-wide identification, characterization, and expression profiling of the legume BZR transcription factor gene family[J]. Front Plant Sci, 2018, 9: 1332.
[44]	Cao X, Khaliq A, Lu S, et al. Genome-wide identification and characterization of the BES1 gene family in apple(Malus domestica)[J]. Plant Biol, 2020, 22(4): 723-733.
[45]	Song XM, Ma X, Li CJ, et al. Comprehensive analyses of the BES1 gene family in Brassica napus and examination of their evolutionary pattern in representative species[J]. BMC Genomics, 2018, 19(1): 346.