金钗石斛bHLH转录因子家族全基因组鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0907

摘要/Abstract

摘要：

【目的】 bHLH转录因子广泛存在于植物中，是调控植物生长发育和响应逆境胁迫的重要转录因子家族之一。从金钗石斛全基因组中鉴定DnbHLHs转录因子家族，分析其表达特点，为后续深入研究DnbHLHs转录因子的生物学功能奠定基础。【方法】 利用生物信息学方法从金钗石斛全基因组中鉴定出bHLH转录因子家族成员，并对其理化特征、系统进化关系、基因结构、染色体定位和启动子顺式作用元件等进行分析，利用转录组数据和RT-qPCR分析DnbHLHs转录因子的组织表达模式和不同非生物胁迫下的表达规律。【结果】 在金钗石斛中共鉴定出137个bHLH转录因子，DnbHLHs蛋白的氨基酸数量为85-1 260，分子量为9 855.23-140 024.19 Da，等电点为4.52-10.66，不稳定系数为31.94-87.42，脂溶指数为56.66-106.06，亲水指数为-0.815-0.185。DnbHLHs分布于金钗石斛的19条染色体上，都存在bHLH结构域，与拟南芥的bHLH同源，且组内、种间存在着共线性关系。基因表达模式分析显示，DnbHLHs家族成员存在组织表达特异性，且DnbHLH26和DnbHLH33对多种逆境胁迫有响应。【结论】 从金钗石斛全基因组中鉴定出137个DnbHLHs转录因子家族成员，该家族成员的理化性质和保守基序存在特异性和多样性的特征，不同DnbHLHs转录因子之间具有组织表达差异性，且DnbHLH26主要受高盐和高温胁迫的诱导，DnbHLH33主要受高盐、高温和低温胁迫的诱导。

关键词: 金钗石斛, 转录因子, bHLH, 生物信息学, 表达模式分析

Abstract:

【Objective】 bHLH transcription factors exist widely in plants and are one of the important transcription factor families that regulate plant growth and development and respond to stress. The DnbHLHs transcription factor family was identified from the whole genome of Dendrobium nobile and its expression characteristics were analyzed, which laid the foundation for further study on the biological function of DnbHLHs transcription factor. 【Method】 The bHLH transcription factor family members were identified from the whole genome of D. nobile by bioinformatics method, and their physical and chemical characteristics, phylogenetic relationship, gene structure, chromosome localization and promoter cis-acting elements were analyzed. Transcriptome data and RT-qPCR were used to analyze the tissue expression patterns of DnbHLHs transcription factors and their expression patterns under different abiotic stresses. 【Result】 A total of 137 bHLH transcription factors were identified in D. nobile. The amino acid number of DnbHLHs protein was 85-1 260, the molecular weight was 9 855.23-140 024.19 Da, the isoelectric point was 4.52-10.66, the instability coefficient was 31.94-87.42, the fat solubility index was 56.66-106.06, and the hydrophilic index was -0.815-0.185. DnbHLHs were distributed on 19 chromosomes of D. nobile, all of which had a bHLH domain and were homologous to the bHLH of Arabidopsis thaliana, and there are collinear relationships between groups and species. Analysis of gene expression patterns showed that DnbHLHs family members had tissue specificity, and DnbHLH26 and DnbHLH33 were responsive to various stresses. 【Conclusion】 A total of 137 DnbHLHs transcription factor family members are identified from the whole genome of D. nobile. The physicochemical properties and conserved mods of the family members showe specificity and diversity. There are tissue expression differences among different DnbHLHs transcription factors, and DnbHLH26 is mainly induced by high salt and high temperature stress. DnbHLH33 is mainly induced by high salt, high temperature and low temperature stress.

Key words: Dendrobium nobile, transcription factor, bHLH, bioinformatics, expression pattern analysis

王健, 杨莎, 孙庆文, 陈宏宇, 杨涛, 黄园. 金钗石斛bHLH转录因子家族全基因组鉴定及表达分析[J]. 生物技术通报, 2024, 40(6): 203-218.

WANG Jian, YANG Sha, SUN Qing-wen, CHEN Hong-yu, YANG Tao, HUANG Yuan. Genome-wide Identification and Expression Analysis of bHLH Transcription Factor Family in Dendrobium nobile[J]. Biotechnology Bulletin, 2024, 40(6): 203-218.

图/表 13

表1 引物序列

Table 1 Primer sequences

基因 Gene	上游引物 Forward primer（5'-3'）	下游引物 Reverse primer（5'-3'）
DnbHLH26	GGTCCTTCGCCATCTTC	CGTCAGTAACTCGGTCAA
DnbHLH33	ACGCAGTCCTTCATCAAC	GTTCATCATCTCCACACTATG
Ubiquitin	CGCCGTCAACCTCATTCCAT	GTGAGGTAGCGACCGTGGC

图1 金钗石斛bHLH家族蛋白系统发育进化树

Fig. 1 Phylogenetic tree of bHLH family protein in D. nobile

图2 金钗石斛bHLH家族蛋白序列多重比对

Fig. 2 Multiple alignment of the protein sequences of D. nobile bHLH family

图3 金钗石斛bHLH转录因子家族保守motif预测

Fig. 3 Conserved motif prediction of bHLH transcription factor family in D. nobile

图4 金钗石斛bHLH转录因子家族蛋白结构域

Fig. 4 Protein domain of D. nobile bHLH transcription factor family

图5 金钗石斛bHLH转录因子家族基因结构

Fig. 5 Gene structure of D. nobile bHLH transcription factor family

图6 金钗石斛bHLH转录因子家族染色体定位

Fig. 6 Chromosome localization of D. nobile bHLH transcription factor family

图7 金钗石斛bHLH转录因子家族成员启动子顺式作用元件

Fig. 7 Promoter cis-acting elements of D. nobile bHLH transcription factor family members

图8 金钗石斛bHLH转录因子家族组内共线性关系

Fig. 8 Collinearity of bHLH transcription factor family groups in D. nobile

图9 金钗石斛、拟南芥、水稻种间共线性关系

Fig. 9 Collinear relationships among D. nobile, Arabidopsis thaliana and Oryza sativa species

图10 金钗石斛bHLH转录因子家族表达模式色阶表示Log2（TPM+1）的值

Fig. 10 Expression patterns of bHLH transcription factor family in D. nobile The level represents the value of Log2（TPM+1）

图11 金钗石斛bHLH转录因子组织表达模式分析

Fig. 11 Tissue expression pattern analysis of bHLH transcription factors in D. nobile

图12 金钗石斛bHLH转录因子胁迫处理表达模式分析

Fig. 12 Analysis of expression patterns of bHLH transcription factors under stress treatment in D. nobile

参考文献 44

[1]	Waseem M, Nkurikiyimfura O, Niyitanga S, et al. GRAS transcription factors emerging regulator in plants growth, development, and multiple stresses[J]. Mol Biol Rep, 2022, 49(10): 9673-9685. doi: 10.1007/s11033-022-07425-x pmid: 35713799
[2]	令狐楚, 谷荣辉, 秦礼康. 金钗石斛的化学成分及药理作用研究进展[J]. 中草药, 2021, 52(24): 7693-7708.
	Linghu C, Gu RH, Qin LK. Research progress on chemical constituents and pharmacological effects of Dendrobium nobile[J]. Chin Tradit Herb Drugs, 2021, 52(24): 7693-7708.
[3]	Zheng SG, Hu YD, Zhao RX, et al. Quantitative assessment of secondary metabolites and cancer cell inhibiting activity by high performance liquid chromatography fingerprinting in Dendrobium nobile[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2020, 1140: 122017.
[4]	Luo AX, He XJ, Zhou SD, et al. Purification, composition analysis and antioxidant activity of the polysaccharides from Dendrobium nobile Lindl[J]. Carbohydr Polym, 2010, 79(4): 1014-1019.
[5]	Nie XQ, Chen Y, Li W, et al. Anti-aging properties of Dendrobium nobile Lindl.: from molecular mechanisms to potential treatments[J]. J Ethnopharmacol, 2020, 257: 112839.
[6]	张明, 夏鸿西, 朱利泉. 石斛组织培养研究进展[J]. 中国中药杂志, 2000, 25(6): 323.
	Zhang M, Xia HX, Zhu LQ. Research progress of Dendrobium tissue culture[J]. China J Chin Mater Med, 2000, 25(6): 323.
[7]	Guo AY, Chen X, Gao G, et al. PlantTFDB: a comprehensive plant transcription factor database[J]. Nucleic Acids Res, 2008, 36(Database issue): D966-D969.
[8]	Atchley WR, Terhalle W, Dress A. Positional dependence, cliques, and predictive motifs in the bHLH protein domain[J]. J Mol Evol, 1999, 48(5): 501-516. doi: 10.1007/pl00006494 pmid: 10198117
[9]	Albert NW, Lewis DH, Zhang HB, et al. Light-induced vegetative anthocyanin pigmentation in Petunia[J]. J Exp Bot, 2009, 60(7): 2191-2202. doi: 10.1093/jxb/erp097 pmid: 19380423
[10]	Lu J, Webb R, Richardson JA, et al. MyoR: a muscle-restricted basic helix-loop-helix transcription factor that antagonizes the actions of MyoD[J]. Proc Natl Acad Sci USA, 1999, 96(2): 552-557. pmid: 9892671
[11]	Heim MA, Jakoby M, Werber M, et al. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity[J]. Mol Biol Evol, 2003, 20(5): 735-747. doi: 10.1093/molbev/msg088 pmid: 12679534
[12]	李洁. 植物转录因子与基因调控[J]. 生物学通报, 2004, 39(3): 9-11.
	Li J. Plant transcription factors and gene regulation[J]. Bull Biol, 2004, 39(3): 9-11.
[13]	Seo JS, Joo J, Kim MJ, et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice[J]. Plant J, 2011, 65(6): 907-921.
[14]	王玉军, 郝宇钧, 戴继勋, 等. OsbHLH1基因在拟南芥中表达及耐低温能力的研究[J]. 高技术通讯, 2004, 14(4): 35-38.
	Wang YJ, Hao YJ, Dai JX, et al. Expression and low temperature tolerance of OsbHLH1 gene in Arabidopsis thaliana[J]. High Technology Communication, 2004, 14(4): 4-12.
[15]	Zhang J, Liu B, Li MS, et al. The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis[J]. Plant Cell, 2015, 27(3): 787-805.
[16]	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
[17]	Cui BL, Huang M, Guo CD, et al. Cloning and expression analysis of DnMSI1 gene in orchid species Dendrobium nobile Lindl[J]. Plant Signal Behav, 2022, 17(1): 2021649.
[18]	郝立冬, 郭海滨, 李明, 等. 玉米MYC基因家族的结构特点和表达模式分析[J]. 基因组学与应用生物学, 2021, 40(4): 1748-1753.
	Hao LD, Guo HB, Li M, et al. Analysis of characteristics and expression pattern of maize MYC gene family[J]. Genom Appl Biol, 2021, 40(4): 1748-1753.
[19]	王力伟, 房永雨, 刘红葵, 等. bHLH转录因子的研究进展[J]. 畜牧与饲料科学, 2020, 41(1): 23-27.
	Wang LW, Fang YY, Liu HK, et al. Research progress of bHLH transcription factors[J]. Anim Husb Feed Sci, 2020, 41(1): 23-27.
[20]	何开平, 吴楚. bHLH转录因子对植物形态发生的影响[J]. 安徽农业科学, 2010, 38(35): 19957-19959.
	He KP, Wu C. The effects of bHLH transcription factors on plant morphogenesis[J]. J Anhui Agric Sci, 2010, 38(35): 19957-19959.
[21]	Zhang TT, Lv W, Zhang HS, et al. Genome-wide analysis of the basic Helix-Loop-Helix(bHLH)transcription factor family in maize[J]. BMC Plant Biol, 2018, 18(1): 235.
[22]	Wei KF, Chen HQ. Comparative functional genomics analysis of bHLH gene family in rice, maize and wheat[J]. BMC Plant Biol, 2018, 18(1): 309.
[23]	徐秀荣, 杨克彬, 王思宁, 等. 毛竹bHLH转录因子的鉴定及其在干旱和盐胁迫条件下的表达分析[J]. 植物科学学报, 2019, 37(5): 610-620.
	Xu XR, Yang KB, Wang SN, et al. Identification of bHLH transcription factors in moso bamboo(Phyllostachys edulis)and their expression analysis under drought and salt stress[J]. Plant Sci J, 2019, 37(5): 610-620.
[24]	Wang Y, Liu AZ. Genomic characterization and expression analysis of basic Helix-loop-Helix(bHLH)family genes in traditional Chinese herb Dendrobium officinale[J]. Plants, 2020, 9(8): 1044.
[25]	Wei S, Xin C, Hui L, et al. Genome-wide identification and analyses of bHLH family genes in Brassica napus[J]. Canadian Journal of Plant Science, 2019, 99(5): 589-598.
[26]	Kavas M, Baloğlu MC, Atabay ES, et al. Genome-wide characterization and expression analysis of common bean bHLH transcription factors in response to excess salt concentration[J]. Mol Genet Genomics, 2016, 291(1): 129-143. doi: 10.1007/s00438-015-1095-6 pmid: 26193947
[27]	Toledo-Ortiz G, Huq E, Quail PH. The Arabidopsis basic/helix-loop-helix transcription factor family[J]. Plant Cell, 2003, 15(8): 1749-1770. doi: 10.1105/tpc.013839 pmid: 12897250
[28]	武家颂, 崔欣茹, 张景荣, 等. 拟南芥bHLH转录因子在根毛发育中的作用[J]. 杭州师范大学学报: 自然科学版, 2022, 21(2): 144-151.
	Wu JS, Cui XR, Zhang JR, et al. Role of bHLH transcription factor members in Arabidopsis root hair development[J]. J Hangzhou Norm Univ Nat Sci Ed, 2022, 21(2): 144-151.
[29]	Liu WW, Tai HH, Li SS, et al. bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism[J]. New Phytol, 2014, 201(4): 1192-1204.
[30]	刘文文, 李文学. 植物bHLH转录因子研究进展[J]. 生物技术进展, 2013, 3(1): 7-11. doi: 10.3969/j.issn.2095-2341.2013.01.02
	Liu WW, Li WX. Progress of plant bHLH transcription factor[J]. Curr Biotechnol, 2013, 3(1): 7-11.
[31]	冯建英, 李立芹, 鲁黎明. 马铃薯bHLH转录因子家族全基因组鉴定与表达分析[J]. 生物技术通报, 2022, 38(2): 21-33. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1327
	Feng JY, Li LQ, Lu LM. Genome-wide identification and expression analysis of the bHLH transcription factor family in Solanum tuberosum[J]. Biotechnol Bull, 2022, 38(2): 21-33.
[32]	Pires N, Dolan L. Origin and diversification of basic-helix-loop-helix proteins in plants[J]. Mol Biol Evol, 2010, 27(4): 862-874. doi: 10.1093/molbev/msp288 pmid: 19942615
[33]	高莉娟, 张正社, 文裕, 等. 象草全基因组bHLH转录因子家族鉴定及表达分析[J]. 草业学报, 2022, 31(3): 47-59. doi: 10.11686/cyxb2020593
	Gao LJ, Zhang ZS, Wen Y, et al. Genome-wide identification and expression analysis of the bHLH transcription factor family in Cenchrus purpureus[J]. Acta Prataculturae Sin, 2022, 31(3): 47-59.
[34]	朱涛, 李芳菲, 杨海涵, 等. 山药bHLH基因家族鉴定及表达分析[J]. 信阳师范学院学报: 自然科学版, 2022, 35(3): 393-399.
	Zhu T, Li FF, Yang HH, et al. Identification and expression ananlysis of bHLH gene family in Chinese yam(Dioscoreae rhizoma)[J]. J Xinyang Norm Univ Nat Sci Ed, 2022, 35(3): 393-399.
[35]	廖桂花, 段钰, 王丛丛, 等. 赪桐bHLH转录因子家族鉴定及生物信息学分析[J/OL]. 分子植物育种, 2023: 1-17. http://kns.cnki.net/kcms/detail/46.1068.S.20230529.1303.022.html.
	Liao GH, Duan Y, Wang CC, et al. Clerod bHLH transcription factor family identification and bioinformatics analysis[J/OL]. Molecular Plant Breeding: 1-17. http://kns.cnki.net/kcms/detail/46.1068.S.20230529.1303.022.html.
[36]	Wang PF, Wang HL, Wang YM, et al. Analysis of bHLH genes from foxtail millet(Setaria italica)and their potential relevance to drought stress[J]. PLoS One, 2018, 13(11): e0207344.
[37]	Fan Y, Lai DL, Yang H, et al. Genome-wide identification and expression analysis of the bHLH transcription factor family and its response to abiotic stress in foxtail millet(Setaria italica L.)[J]. BMC Genomics, 2021, 22(1): 778.
[38]	Zhan H, Liu HZ, Ai WF, et al. Genome-wide identification and expression analysis of the bHLH transcription factor family and its response to abiotic stress in Mongolian oak(Quercus mongolica)[J]. Curr Issues Mol Biol, 2023, 45(2): 1127-1148. doi: 10.3390/cimb45020075 pmid: 36826020
[39]	Muhammad H K, Xuemei F, Huabo W, et al. Genome-wide identification and expression analysis of the bHLH transcription factor family in wintersweet(Chimonanthus praecox)[J]. International Journal of Molecular Sciences, 2023, 24(17): 13461.
[40]	陈媞颖, 刘娟, 袁媛, 等. 黄芩bHLH转录因子基因家族生物信息学及表达分析[J]. 中草药, 2018, 49(3): 671-677.
	Chen TY, Liu J, Yuan Y, et al. Analysis of bioinformatics and expression level of bHLH transcription factors in Scutellaria baicalensis[J]. Chin Tradit Herb Drugs, 2018, 49(3): 671-677.
[41]	何昌文, 朱丽, 沈珊, 等. 银杏bHLH91转录因子基因的克隆及表达分析[J]. 广西植物, 2018, 38(2): 202-209.
	He CW, Zhu L, Shen S, et al. Cloning and expression analysis of a bHLH91 transcription factor gene from Ginkgo biloba[J]. Guihaia, 2018, 38(2): 202-209.
[42]	孟富宣, 杨玉皎, 段元杰, 等. 甜橙bHLH转录因子家族的鉴定及生物信息学分析[J]. 西南林业大学学报: 自然科学, 2020, 40(5): 73-86.
	Meng FX, Yang YJ, Duan YJ, et al. Identification and bioinformatics analysis of the bHLH transcription factor family in Citrus sinensis[J]. J Southwest For Univ Nat Sci, 2020, 40(5): 73-86.
[43]	Guo JR, Sun BX, He HR, et al. Current understanding of bHLH transcription factors in plant abiotic stress tolerance[J]. Int J Mol Sci, 2021, 22(9): 4921.
[44]	Chinnusamy V, Ohta M, Kanrar S, et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis[J]. Genes Dev, 2003, 17(8): 1043-1054.