Cloning and Expression of DcbHLH14 from Dendrobium catenatum Lindl.
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摘要:目的 克隆铁皮石斛转录因子DcbHLH14基因并分析其在非生物胁迫响应中的表达情况，为研究DcbHLH14基因的功能提供理论参考。方法 通过同源克隆从铁皮石斛叶组织中得到DcbHLH14基因，对其编码的蛋白序列特征和组织表达特性进行分析，并采用qRT-PCR对DcbHLH14基因在低温、干旱和ABA处理过程中的表达量进行分析。结果 DcbHLH14基因开放阅读框（Open reading frame, ORF）为1269 bp，与参考序列存在7个碱基差异，仅含有1个外显子且无内含子，编码422个氨基酸。DcbHLH14蛋白的分子式为C2011H3192N586O613S13，理论分子量为45.8 kD，理论等电点（pI）为5.98，含有bHLH家族保守结构域bHLH-MYC-N和HLH，属于bHLH家族，与鼓槌石斛（Dendrobium chrysotoxum）和春兰（Cymbidium goeringii）bHLH蛋白的氨基酸序列同源性较高，分别为97.16%和86.90%。转录组分析结果显示，DcbHLH14基因在云南产地野生铁皮石斛花蕾中的表达量最高，在叶中的表达量最低。进一步qRT-PCR分析结果表明，该基因在广东丹霞铁皮石斛叶中的表达量最高，而在茎中的表达量最低。DcbHLH14基因启动子富含多种与水分胁迫、低温、脱水以及ABA响应等相关的顺式作用元件。DcbHLH14基因明显受到低温、干旱和ABA诱导，低温和ABA处理6 h后DcbHLH14表达量被显著提高并达到峰值，分别是处理前的12.6倍和3.7倍；干旱处理9 h后，DcbHLH14表达量最高，是处理前的6.5倍，达到极显著差异水平。结论 DcbHLH14基因可能在转录水平上通过依赖于ABA信号通路途径响应低温和干旱胁迫，从而调控下游功能基因表达，提高铁皮石斛抗逆性。
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关键词:
- 铁皮石斛 /
- DcbHLH14基因 /
- 非生物胁迫 /
- 表达分析
Abstract:Objective Functions of DcbHLH14, a basic helix-loop-helix transcription factor of Dendrobium catenatum Lindl., in response to abiotic stresses were studied.Method DcbHLH14 was cloned from D. catenatum leaves using homologous cloning method for a bioinformatic analysis on the gene and expression in tissues. Gene expressions under low temperature, drought, and abscisic acid (ABA) stresses were determined.Result The ORF of DcbHLH14 was 1 269 bp and encoded 422 amino acids. Itcontained one exon, no intron, and 7 bases that were different from the reference sequences. The theoretical molecular weight was 45.8 kD, isoelectric point pH 5.98, and molecular formula C2011H3192N586O613S13. Its conserved domains contained bHLH-MYC-N and HLH proteins that had high similarities with the bHLH proteins in D. chrysotoxum at 97.16% and in Cymbidium goeringii at 86.90%. The transcriptome analysis revealed high expressions in the flower buds and columns but low in the leaves of the wild D. catenatum from Yunnan, whereas the qRT-PCR analysis showed high expressions in the leaves and low in the stems of the sample from Danxia, Guangdong. The promoters of DcbHLH14 contained numerous cis-acting elements associated with the responses to water-depletion, low temperature, dehydration, and ABA stresses, which significantly affected expression of the gene. For instance, DcbHLH14 was upregulated to peak in 6 h after a low temperature or ABA treatment reaching 12.6 or 3.7 times, respectively, as well as by a 9 h drought stress to become as high as 6.5 times of control. Conclusion It was postulated that DcbHLH14 responded to low temperature or drought stress through the ABA signaling pathway at transcription level. Hence, the tolerance of D. catenatum to the abiotic stresses could be manipulated by regulating the expression of the downstream functional gene.-
Keywords:
- Dendrobium catenatum /
- DcbHLH14 /
- abiotic stress /
- expression analysis
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图 1 DcbHLH14基因克隆（A）及染色体定位（B）结果
M：Marker；1：DcbHLH14基因 cDNA 电泳。
Figure 1. DcbHLH14 cloning (A) and chromosome locating (B)
M: Marker; 1: cDNA electrophoresis of DcbHLH14.
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图 2 DcbHLH14基因cDNA序列及其推导氨基酸序列分析
红色箭头表示DcbHLH14基因与参考序列间存在的碱基差异。
Figure 2. cDNA and deduced amino acid sequences of DcbHLH14
Red arrow represents difference on base between DcbHLH14 and reference sequences.
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图 4 DcbHLH14蛋白亲疏水分析（A）、二级结构预测（B）及三级结构预测（C）
Figure 4. Hydrophilicity and hydrophobicity of DcbHLH14 protein (A), predicted secondary structure (B), and tertiary structure (C)
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图 5 DcbHLH14蛋白与其他物种bHLH蛋白的多重序列比对分析结果
实线表示bHLH-MYC-N结构域；虚线表示HLH结构域。
Figure 5. Multiple sequence alignment between DcbHLH14 and bHLH proteins from other plants
Solid line represents bHLH-MYC-N domain; dashed line, HLH domain.
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图 6 基于不同物种bHLH蛋白氨基酸序列构建的系统发育进化树
Figure 6. Phylogenetic tree based on amino acid sequences in bHLH protein of different species
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图 8 DcbHLH14基因组织表达特性的qRT-PCR分析
Figure 8. qRT-PCR analysis on DcbHLH14 expressions in tissues
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图 9 DcbHLH14基因在低温（A）、干旱（B）和ABA（C）处理下的表达情况
*表示与0 h差异显著（P＜0.05）；***表示与0 h极显著差异（P＜0.001）；
Figure 9. Expressions of DcbHLH14 under cold stress (A), drought stress (B), and ABA treatment (C)
* represents significant difference from control (0 h) at P＜0.05; *** represents extremely significant difference from control (0 h) at P＜0.001.
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图 10 DcbHLH14基因在低温、干旱和ABA处理下表达水平的qRT-PCR分析结果
不同小写字母表示同一处理不同时间之间差异显著（P<0.05）。
Figure 10. qRT-PCR analysis on expressions of DcbHLH14
under low temperature, drought, and ABA treatment Different lowercase letters indicant significant difference among different treatment times of the same treatment (P<0.05).
表 1 DcbHLH14基因启动子顺式作用元件分析结果
Table 1 Cis-acting elements in DcbHLH14 promoter
序号 No. 元件名称 Element 元件序列 Element sequence 数目 Number 功能预测 Predicted function 1 MYCCONSENSUSAT CANNTG 18 低温响应 Low temperature responsive 2 MYBCORE CNGTTR 4 水分胁迫调控 Regulation of water stress 3 MYB2CONSENSUSAT YAACKG 3 脱水响应 Dehydration responsive 4 LTRECOREATCOR15 CCGAC 3 低温响应 Low temperature responsive 5 MYBATRD22 CTAACCA 2 脱水响应 Dehydration responsive 6 MYB1AT WAACCA 1 脱水响应 Dehydration responsive 7 ABRELATERD1 ACGTG 1 ABA响应 Abscisic acid responsive 
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[1] 蔡琳, 彭鹏. 名贵中药铁皮石斛化学成分及其药理作用浅述 [J]. 安徽化工, 2021, 47(1):24−25. DOI: 10.3969/j.issn.1008-553X.2021.01.008 CAI L, PENG P. A brief introduction on the chemical constituents and pharmacological action of rare Chinese medicine Dendrobium officinale [J]. Anhui Chemical Industry, 2021, 47(1): 24−25.(in Chinese) DOI: 10.3969/j.issn.1008-553X.2021.01.008
[2] 黄嘉雯, 陈小阳, 刘涛利, 等. 花色素苷合成关键调节基因的研究进展 [J]. 分子植物育种, 2019, 17(11):3602−3608. DOI: 10.13271/j.mpb.017.003602 HUANG J W, CHEN X Y, LIU T L, et al. Research progress of the key regulatory genes for anthocyanin synthesis [J]. Molecular Plant Breeding, 2019, 17(11): 3602−3608.(in Chinese) DOI: 10.13271/j.mpb.017.003602
[3] 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. DOI: 10.12160/j.issn.1672-5190.2020.01.005 WANG L W, FANG Y Y, LIU H K, et al. Research progress of bHLH transcription factors [J]. Animal Husbandry and Feed Science, 2020, 41(1): 23−27.(in Chinese) DOI: 10.12160/j.issn.1672-5190.2020.01.005
[4] 陈清, 汤浩茹, 董晓莉, 等. 植物Myb转录因子的研究进展 [J]. 基因组学与应用生物学, 2009, 28(2):365−372. CHEN Q, TANG H R, DONG X L, et al. Progress in the study of plant myb transcription factors [J]. Genomics and Applied Biology, 2009, 28(2): 365−372.(in Chinese)
[5] AMOUTZIAS G, VERON A, WEINER J, et al. One billion years of bZIP transcription factor evolution: Conservation and change in dimerization and DNA-binding site specificity [J]. Molecular Biology and Evolution, 2006, 24(3): 827−835. DOI: 10.1093/molbev/msl211
[6] 覃超. 甜瓜CmbHLH93和CmbHLH130基因在果实发育中的作用[D]. 呼和浩特: 内蒙古大学, 2020: 84－86. QIN C. The role of CmbHLH93 and CmbHLH130 in fruit development of melon[D]. Hohhot: Inner Mongolia University, 2020: 84-86. (in Chinese)
[7] 张娇. 两个bHLH转录因子(AtLPl和AtLP2)在拟南芥细胞伸长生长中的功能研究[D]. 武汉: 华中师范大学, 2019: 28－29. ZHANG J. Study on the roles of two bHLH transcription factors(AtLPl and AtLP2)in cell elongation of Arabidopsis thaliana[D]. Wuhan: Central China Normal University, 2019: 28－29. (in Chinese)
[8] LIU Y, LI X, LI K, et al. Multiple bHLH proteins form heterodimers to mediate CRY2-dependent regulation of flowering-time in Arabidopsis [J]. PLoS Genetics, 2013, 9(10): e1003861. DOI: 10.1371/journal.pgen.1003861
[9] 宋建辉. bHLH113调控拟南芥开花和花青素合成的分子机制研究[D]. 杭州: 浙江农林大学, 2020. SONG J H. The molecular regulatory mechanism of flowering and anthocyanin by bHLH113 in Arabidopsis[D]. Hangzhou: Zhejiang A & F University, 2020. (in Chinese)
[10] WU H H, REN Z Y, ZHENG L, et al. The bHLH transcription factor GhPAS1 mediates BR signaling to regulate plant development and architecture in cotton [J]. The Crop Journal, 2021, 9(5): 1049−1059. DOI: 10.1016/j.cj.2020.10.014
[11] ZHANG J H, LV H Z, LIU W J, et al. bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza [J]. Chinese Herbal Medicines, 2020, 12(3): 237−246. DOI: 10.1016/j.chmed.2020.04.001
[12] MENG F W, YANG C, CAO J D, et al. A bHLH transcription activator regulates defense signaling by nucleo-cytosolic trafficking in rice [J]. Journal of Integrative Plant Biology, 2020, 62(10): 1552−1573. DOI: 10.1111/jipb.12922
[13] 尹航. 露地菊CgbHLH113基因的克隆及功能分析[D]. 哈尔滨: 东北林业大学, 2021. YIN H. Cloning and functional analysis of CgbHLH113 gene from Chrysanthemum × grandiflora[D]. Harbin: Northeast Forestry University, 2021. (in Chinese)
[14] LI Y Y, SUI X Y, YANG J S, et al. A novel bHLH transcription factor, NtbHLH1, modulates iron homeostasis in tobacco (Nicotiana tabacum L.) [J]. Biochemical and Biophysical Research Communications, 2020, 522(1): 233−239. DOI: 10.1016/j.bbrc.2019.11.063
[15] YI K K, WU Z C, ZHOU J, et al. OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice [J]. Plant Physiology, 2005, 138(4): 2087−2096. DOI: 10.1104/pp.105.063115
[16] WANG F B, ZHU H, CHEN D H, et al. A grape bHLH transcription factor gene, VvbHLH1, increases the accumulation of flavonoids and enhances salt and drought tolerance in transgenic Arabidopsis thaliana [J]. Plant Cell, Tissue and Organ Culture (PCTOC), 2016, 125(2): 387−398. DOI: 10.1007/s11240-016-0953-1
[17] CHEN H C, CHENG W H, HONG C Y, et al. The transcription factor OsbHLH035 mediates seed germination and enables seedling recovery from salt stress through ABA-dependent and ABA-independent pathways, respectively [J]. Rice, 2018, 11(1): 50. DOI: 10.1186/s12284-018-0244-z
[18] GAO Y, WU M Q, ZHANG M J, et al. Roles of a maize phytochrome-interacting factors protein ZmPIF3 in regulation of drought stress responses by controlling stomatal closure in transgenic rice without yield penalty [J]. Plant Molecular Biology, 2018, 97(4): 311−323.
[19] REN Y R, YANG Y Y, ZHAO Q, et al. MdCIB1, an apple bHLH transcription factor, plays a positive regulator in response to drought stress [J]. Environmental and Experimental Botany, 2021, 188: 104523. DOI: 10.1016/j.envexpbot.2021.104523
[20] ZHAO Q, XIANG X H, LIU D, et al. Tobacco transcription factor NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis [J]. Frontiers in Plant Science, 2018, 9: 381. DOI: 10.3389/fpls.2018.00381
[21] DONG H Z, CHEN Q M, DAI Y Q, et al. Genome-wide identification of PbrbHLH family genes, and expression analysis in response to drought and cold stresses in pear (Pyrus bretschneideri) [J]. BMC Plant Biology, 2021, 21(1): 86. DOI: 10.1186/s12870-021-02862-5
[22] JIN R, KIM H S, YU T, et al. Identification and function analysis of bHLH genes in response to cold stress in sweetpotato [J]. Plant Physiology and Biochemistry, 2021, 169: 224−235. DOI: 10.1016/j.plaphy.2021.11.027
[23] YU Z M, ZHANG G H, TEIXEIRA DA SILVA J A, et al. The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development [J]. Plant Science, 2021, 309: 110952. DOI: 10.1016/j.plantsci.2021.110952
[24] CHEN Y, WANG Y Z, LYU P, et al. Comparative transcriptomic analysis reveal the regulation mechanism underlying MeJA-induced accumulation of alkaloids in Dendrobium officinale [J]. Journal of Plant Research, 2019, 132(3): 419−429. DOI: 10.1007/s10265-019-01099-6
[25] 张志勇, 阳静, 齐泽民. 铁皮石斛总RNA提取方法的比较研究 [J]. 江苏农业科学, 2017, 45(4):33−35. DOI: 10.15889/j.issn.1002-1302.2017.04.009 ZHANG Z Y, YANG J, QI Z M. Comparative study on extraction methods of total RNA from Dendrobium candidum [J]. Jiangsu Agricultural Sciences, 2017, 45(4): 33−35.(in Chinese) DOI: 10.15889/j.issn.1002-1302.2017.04.009
[26] ZHANG G Q, LIU K W, LI Z, et al. The Apostasia genome and the evolution of orchids [J]. Nature, 2017, 549(7672): 379−383. DOI: 10.1038/nature23897
[27] WANG Y, LIU A Z. Genomic characterization and expression analysis of basic Helix-loop-Helix (bHLH) family genes in traditional Chinese herb Dendrobium officinale [J]. Plants (Basel, Switzerland), 2020, 9(8): 1044.
[28] 李季生, 李娜, 贾漫丽, 等. 基于转录组数据挖掘桑树bHLH转录因子家族 [J]. 分子植物育种, 2022, 20(6):1798−1810. DOI: 10.13271/j.mpb.020.001798 LI J S, LI N, JIA M L, et al. Mining bHLH transcription factor family of mulberry based on transcriptome data [J]. Molecular Plant Breeding, 2022, 20(6): 1798−1810.(in Chinese) DOI: 10.13271/j.mpb.020.001798
[29] 王菊萍, 王珍, 张铁军, 等. 蒺藜苜蓿MtbHLH148转录因子的克隆与转化及其功能分析 [J]. 西北植物学报, 2019, 39(6):963−973. DOI: 10.7606/j.issn.1000-4025.2019.06.0963 WANG J P, WANG Z, ZHANG T J, et al. Cloning and analysis of a basic Helix-loop-Helix (bHLH) transcription factor MtbHLH148 from Medicago truncatula L [J]. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39(6): 963−973.(in Chinese) DOI: 10.7606/j.issn.1000-4025.2019.06.0963
[30] SUN W J, JIN X, MA Z T, et al. Basic helix-loop-helix (bHLH) gene family in Tartary buckwheat (Fagopyrum tataricum): Genome-wide identification, phylogeny, evolutionary expansion and expression analyses [J]. International Journal of Biological Macromolecules, 2020, 155: 1478−1490. DOI: 10.1016/j.ijbiomac.2019.11.126
[31] 杨贞, 蔡友铭, 张永春, 等. 基于SRAP分子标记的铁皮石斛遗传多样性分析 [J]. 上海农业学报, 2019, 35(5):23−27. DOI: 10.15955/j.issn1000-3924.2019.05.05 YANG Z, CAI Y M, ZHANG Y C, et al. Genetic diversity analysis of Dendrobium officinale based on SRAP molecular markers [J]. Acta Agriculturae Shanghai, 2019, 35(5): 23−27.(in Chinese) DOI: 10.15955/j.issn1000-3924.2019.05.05
[32] 朱璐璐, 周波. bHLH蛋白在植物发育及非生物胁迫中的调控[J/OL]. 分子植物育种, 2021: 1-14. (2021-02-23). https://kns.cnki.net/kcms/detail/46.1068.S.20210222.1744.012.html. ZHU L L, ZHOU B. Regulation of bHLH protein in plant development and abiotic stress[J/OL]. Molecular Plant Breeding, 2021: 1-14. (2021-02-23). https://kns.cnki.net/kcms/detail/46.1068.S.20210222.1744.012.html.(in Chinese)
[33] CASTILHOS G, LAZZAROTTO F, SPAGNOLO-FONINI L, et al. Possible roles of basic helix-loop-helix transcription factors in adaptation to drought [J]. Plant Science, 2014, 223: 1−7. DOI: 10.1016/j.plantsci.2014.02.010
[34] LIU Y J, JI X Y, NIE X G, et al. Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs [J]. The New Phytologist, 2015, 207(3): 692−709. DOI: 10.1111/nph.13387
[35] JI X Y, NIE X G, LIU Y J, et al. A bHLH gene from Tamarix hispida improves abiotic stress tolerance by enhancing osmotic potential and decreasing reactive oxygen species accumulation [J]. Tree Physiology, 2016, 36(2): 193−207.
[36] 耿晶晶. 甜橙bHLH家族转录因子发掘及CsbHLH18抗寒功能鉴定与作用机制解析[D]. 武汉: 华中农业大学, 2018: 77-79. GENG J J. Genome-wide identification of bHLH transcription factor family in sweet orange(Citrus sinensis) and functional characterization and mechanism analysis of CsbHLH18 in cold resistance[D]. Wuhan: Huazhong Agricultural University, 2018: 77-79. (in Chinese)
[37] PARK S, LEE C, DOHERTY C J, et al. Regulation of the Arabidopsis CBF regulon by a complex low-temperature regulatory network [J]. The Plant Journal, 2015, 82(2): 193−207. DOI: 10.1111/tpj.12796
[38] CHINNUSAMY V, ZHU J K, SUNKAR R. Gene regulation during cold stress acclimation in plants [J]. Methods in Molecular Biology (Clifton, N J), 2010, 639: 39−55.
[39] CHINNUSAMY V, OHTA M, KANRAR S, et al. ICE1: A regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis [J]. Genes & Development, 2003, 17(8): 1043−1054.
[40] SHI Y T, DING Y L, YANG S H. Molecular regulation of CBF signaling in cold acclimation [J]. Trends in Plant Science, 2018, 23(7): 623−637. DOI: 10.1016/j.tplants.2018.04.002
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