小麦组蛋白甲基化酶在杂交种中干旱胁迫表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1524

摘要/Abstract

摘要：

旨在筛选参与小麦杂交种干旱胁迫的组蛋白甲基化酶基因，为二系杂交小麦抗逆性改良提供功能标记。通过网站公开数据库和比较基因组学策略对小麦TaHMT家族175个基因进行筛选获得8个受干旱胁迫诱导表达候选基因（TaHMT21、TaHMT24、TaHMT31、TaHMT42、TaHMT49、TaHMT105、TaHMT143、TaHMT157）。采用生物信息学方法对8个TaHMTs理化性质、蛋白结构、系统发育关系、基因结构和保守基序等进行了系统分析。进一步利用干旱处理的小麦杂交组合（BS278*09YH91-5及父母本）为试材，对8个组蛋白甲基化酶候选基因进行荧光定量PCR验证，发现它们受不同程度干旱胁迫诱导表达，且随着干旱胁迫时间延长基因表达水平呈先上调后下调的模式，其中TaHMT21、TaHMT24、TaHMT42基因表达与杂交组合的抗旱表型相符合，从而推测这些基因可能参与调控杂交种的抗旱性。上述结果为深入研究小麦TaHMT家族基因在杂交种抗旱性中发挥的功能提供了重要线索，同时也为小麦分子育种提供优异抗逆基因资源。

关键词: 小麦, 杂交种, TaHMT, 生物信息学分析, 干旱胁迫

Abstract:

The aim of this study is to screen the histone methyltransferases involved in the drought stress of wheat hybrids，and to provide functional markers for the improvement of stress resistance of two-line hybrid wheat. Screening of the total 175 TaHMT family genes in the wheat through the public databases on the website and the comparative genomics strategies，we obtained 8 candidate histone methyltransferases genes induced by drought stress（TaHMT21，TaHMT24，TaHMT31，TaHMT42，TaHMT49，TaHMT105，TaHMT143，TaHMT157）. The physical and chemical properties，protein structures，phylogenetic relationships，gene structures and conserved motifs of 8 TaHMTs were systematically analyzed via bioinformatics. Using drought treated hybrid wheat combinations（BS278*09YH91-5 and parental）as experimental materials，the candidate genes of 8 histone methyltransferase were verified via quantitative fluorescent PCR. It was found that their expressions were induced by drought stress，and the expression pattern presented mode of up-regulation first and then down-regulation with the drought stress time prolonged. The expressions of gene TaHMT21，TaHMT24 and TaHMT42 accorded with the phenotypes of hybrid combinations，indicating that they may regulate the drought resistance of hybrids. These results provide important clues to deeply study the function of wheat TaHMT family genes in the drought resistance of hybrids as well as also provide excellent stress resistance gene resources for wheat molecular breeding.

Key words: wheat, hybrid, TaHMT, bioinformatics analysis, drought stress

陈佳敏, 刘永杰, 马锦绣, 李丹, 公杰, 赵昌平, 耿洪伟, 高世庆. 小麦组蛋白甲基化酶在杂交种中干旱胁迫表达模式分析[J]. 生物技术通报, 2022, 38(7): 51-61.

CHEN Jia-min, LIU Yong-jie, MA Jin-xiu, LI Dan, GONG Jie, ZHAO Chang-ping, GENG Hong-wei, GAO Shi-qing. Expression Pattern Analysis of Histone Methyltransferase Under Drought Stress in Hybrid Wheat[J]. Biotechnology Bulletin, 2022, 38(7): 51-61.

图/表 9

表1 实时定量PCR引物序列

Table 1 Primer sequences for real-time quantitative PCR

引物名称Primer name	上游引物序列 Forward primer sequence（5'-3'）	下游引物序列 Reverse primer sequence（5'-3'）
actin	GTTGGTGATGAGGCCCAATC	GTGCTACACGGAGCTCATTG
TaHMT21	TGCCGCTGTGGTGTATACTG	CAGCCAAAAGACCCCATCCA
TaHMT24	TAAGGCTAGGGGCAACTCCT	CCATCCAACTCAGCAGTCGT
TaHMT31	GTTTGGTGGTGTGATGGTGC	TGCTGGAATGGTGTCCTTCG
TaHMT42	GGTCTGCGCAAGCAATTGAA	TCGTACAGGGCGTCGATTTC
TaHMT49	TTCTTCACGAGCAGGAAGGT	TTCCTCGGCAGTACTTGCTT
TaHMT105	AGACCTTAGAGAGCGGCGTA	ATGCTCATCCCCGAGAACAC
TaHMT143	TGATTGCAGCATGGACCAGT	GCAAACAGCCCCAAAGTACG
TaHMT157	CTACGGTTGCATCAAGCTCA	GCATCTCACAGTCCTCGTCA

图1 小麦TaHMT候选基因干旱胁迫表达热图 A：在Wheat Expression Brower数据库和WheatOmics数据库上差异表达基因与同源基因的韦恩图;B：8个候选基因在Wheat Expression Brower数据库的表达热图;C：8个候选基因在WheatOmics数据库的表达热图。红色字体为候选基因

Fig.1 Heatmap of TaHMT candidate gene expressions under drought stress in wheat A：Venn diagram of differentially expressed genes and homologous genes on Wheat Expression Brower database and WheatOmics database. B：Expression heatmap of 8 candidate genes in Wheat Expression Brower database. C：Expression heatmap of 8 candidate genes in WheatOmics database. The red font is the candidate gene

表2 TaHMT干旱胁迫候选基因基本信息

Table 2 Basic information of TaHMT drought stress candidate genes

基因名称Gene name	TaHMT21	TaHMT24	TaHMT31	TaHMT42	TaHMT49	TaHMT105	TaHMT143	TaHMT157
基因ID号Gene ID	TraesCS2A02G302100	TraesCS2A02G457100	TraesCS2B02G317900	TraesCS2D02G300800	TraesCS2D02G566400	TraesCS5B02G163000	TraesCS7B02G262900	TraesCS2A02G147100
染色体位置Chromosome location	2A：517 803 993-517 808 473（+）	2A：705 740 079-705 742 875（+）	2B：453 747 757-453 752 627（+）	2D：383 285 805-383 290 377（+）	2D：635 593 374-635 597 307（-）	5B：300 380 740-300 397 790（-）	7B：483 766 747-483 769 174（-）	2A：93 926 568-93 930 236（-）
亚细胞定位Subcellular localization	Nucleus	Nucleus	Nucleus	Nucleus	Nucleus	Nucleus	Nucleus	Nucleus
氨基酸数目Number of amino acids/aa	502	476	529	502	737	1086	390	601
分子量Molecular weight/kD	56 612.99	53 938.51	59 323.00	56 560.96	80 411.70	122 007.19	42 293.23	65 280.55
理论等电点Theoretical pI	4.77	5.47	4.78	4.80	7.09	7.59	4.44	4.48
负电荷残基数（Asp+Glu） Number of negatively charged residues（Asp+Glu）	83	64	84	82	84	146	65	99
正电荷残基数（Arg+Lys） Number of positively charged residues（Arg+Lys）	53	53	53	53	83	148	30	50
总原子数Total number of atoms	7 801	7 540	8 162	7 794	11 137	------	5 789	8 992
分子式Formula	C2458H3830N684O 805S24	C2392H3751N657O 716S24	C2572H4003N717O 843S27	C2458H3826N684O 802S24	C3478H5503N1035O 1080S41	------	C1833H2830N506O 594S26	C2859H4408N762O 937S26
不稳定指数Instability index	51.39	43.26	49.28	53.18	49.44	54.44	54.59	38.91
脂溶指数Aliphatic index	71.14	90.82	71.02	71.53	68.41	76.19	75.95	75.61
亲水性GRAVY	-0.631	-0.219	-0.584	-0.608	-0.511	-0.514	-0.301	-0.285

图2 TaHMT蛋白亲疏水性分析

Fig.2 Analyzed hydrophilicity and hydrophobicity for TaHMT protein

表3 小麦TaHMT蛋白的二级结构分析

Table 3 Secondary structure analysis of TaHMT proteins

图3 TaHMT蛋白的系统进化分析

Fig.3 Phylogenetic analysis of TaHMT

图4 小麦TaHMT干旱胁迫候选基因基因结构和保守基序分析

Fig.4 Gene structure and conserved motifs analysis of wheat TaHMT drought stress candidate genes

图5 TaHMT干旱胁迫候选基因干旱响应元件种类和数目

Fig.5 Types and number of drought response elements of TaHMT drought stress candidate genes

图6 TaHMT干旱胁迫下的表达模式分析 A：营养生长期小麦叶片中基因的表达;B：苗期小麦地上部中基因的表达;C：苗期小麦地下部中基因的表达。图中的误差线表示标准偏差。小写字母表示同一时间不同材料之间的显著差异（P<0.05）

Fig.6 Expressions of TaHMT under drought stress A：Gene expression in the wheat leaves during vegetative growth. B：Gene expression in the shoot of wheat at seedling stage. C：Gene expression in the underground part of wheat at seedling stage. The error line in the figure refers to the standard deviation. The lowercase letters indicate significant differences between different materials at the same time（P<0.05）

参考文献 44

[1]	Goldberg AD, Allis CD, Bernstein E. Epigenetics:a landscape takes shape[J]. Cell, 2007, 128(4):635-638.
[2]	Huang H, Sabari BR, Garcia BA, et al. SnapShot:histone modifications[J]. Cell, 2014, 159(2):458-458. e1.
[3]	Lawrence M, Daujat S, Schneider R. Lateral thinking:how histone modifications regulate gene expression[J]. Trends Genet, 2016, 32(1):42-56.
[4]	Jenuwein T, Allis CD. Translating the histone code[J]. Science, 2001, 293(5532):1074-1080.
[5]	Strahl BD, Allis CD. The language of covalent histone modifications[J]. Nature, 2000, 403(6765):41-45.
[6]	Feng Q, Wang HB, Ng HH, et al. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain[J]. Curr Biol, 2002, 12(12):1052-1058.
[7]	Yeates TO. Structures of SET domain proteins:protein lysine methyltransferases make their mark[J]. Cell, 2002, 111(1):5-7.
[8]	Ng DWK, Wang T, Chandrasekharan MB, et al. Plant SET domain-containing proteins:structure, function and regulation[J]. Biochim Biophys Acta, 2007, 1769(5/6):316-329.
[9]	Ahmad A, Dong YZ, Cao XF. Characterization of the PRMT gene family in rice reveals conservation of arginine methylation[J]. PLoS One, 2011, 6(8):e22664.
[10]	Gary JD, Clarke S. RNA and protein interactions modulated by protein arginine methylation[J]. Prog Nucleic Acid Res Mol Biol, 1998, 61:65-131.
[11]	Boisvert FM, Chénard CA, Richard S. Protein interfaces in signaling regulated by arginine methylation[J]. Sci STKE, 2005, 2005(271):re2.
[12]	Bedford MT, Clarke SG. Protein arginine methylation in mammals:who, what, and why[J]. Mol Cell, 2009, 33(1):1-13.
[13]	Chinnusamy V, Zhu JK. Epigenetic regulation of stress responses in plants[J]. Curr Opin Plant Biol, 2009, 12(2):133-139.
[14]	Luo M, Liu XC, Singh P, et al. Chromatin modifications and remodeling in plant abiotic stress responses[J]. Biochim Biophys Acta, 2012, 1819(2):129-136.
[15]	Kumar SV, Wigge PA. H2A. Z-containing nucleosomes mediate the thermosensory response in Arabidopsis[J]. Cell, 2010, 140(1):136-147.
[16]	Ding Y, Lapko H, Ndamukong I, et al. The Arabidopsis chromatin modifier ATX1, the myotubularin-like AtMTM and the response to drought[J]. Plant Signal Behav, 2009, 4(11):1049-1058.
[17]	Chen K, Du KX, Shi YC, et al. H3K36 methyltransferase SDG708 enhances drought tolerance by promoting abscisic acid biosynthesis in rice[J]. New Phytol, 2021, 230(5):1967-1984.
[18]	Sun XW, Chen L, et al. Histone methyltransferase SDG8 in dehydr-ation stress[J]. J Univ Sci Technol China, 2021, 51(2):140-146.
[19]	赵燕昊, 曹跃芬, 孙威怡, 等. 小麦抗旱研究进展[J]. 植物生理学报, 2016, 52(12):1795-1803.
	Zhao YH, Cao YF, Sun WY, et al. The research advances in drought resistance in wheat[J]. Plant Physiol J, 2016, 52(12):1795-1803.
[20]	杨梅. 普通小麦转TaEBP基因系抗旱特性研究[D]. 杨凌: 西北农林科技大学, 2012.
	Yang M. Study on drought-resistance of transgenic wheat with TaEBP gene[D]. Yangling: Northwest A & F University, 2012.
[21]	Edae EA, Byrne PF, et al. Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes[J]. Theor Appl Genet, 2014, 127(4):791-807.
[22]	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.
[23]	Gasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPASy server[M]// Walker JM. The Proteomics Protocols Handbook. Totowa, NJ: Humana Press, 2005:571-607.
[24]	Chou KC, Shen HB. Cell-PLoc:a package of Web servers for predicting subcellular localization of proteins in various organisms[J]. Nat Protoc, 2008, 3(2):153-162.
[25]	Geourjon C, Deléage G. SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Comput Appl Biosci, 1995, 11(6):681-684.
[26]	Kelley LA, Mezulis S, Yates CM, et al. The Phyre2 web portal for protein modeling, prediction and analysis[J]. Nat Protoc, 2015, 10(6):845-858.
[27]	Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers[J]. Proc Int Conf Intell Syst Mol Biol, 1994, 2:28-36.
[28]	Kumar S, Stecher G, Li M, et al. MEGA X:molecular evolutionary genetics analysis across computing platforms[J]. Mol Biol Evol, 2018, 35(6):1547-1549.
[29]	He ZL, Zhang HK, Gao SH, et al. Evolview v2:an online visualization and management tool for customized and annotated phylogenetic trees[J]. Nucleic Acids Res, 2016, 44(W1):W236-W241.
[30]	Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Res, 2002, 30(1):325-327.
[31]	Ding Y, Avramova Z, Fromm M. The Arabidopsis trithorax-like factor ATX1 functions in dehydration stress responses via ABA-dependent and ABA-independent pathways[J]. Plant J, 2011, 66(5):735-744.
[32]	Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat[J]. Front Plant Sci, 2014, 5:170.
[33]	Cao XY, Costa LM, Biderre-Petit C, et al. Abscisic acid and stress signals induce viviparous1 expression in seed and vegetative tissues of maize[J]. Plant Physiol, 2007, 143(2):720-731.
[34]	Yang WT, Baek D, Yun DJ, et al. Rice OsMYB5P improves plant phosphate acquisition by regulation of phosphate transporter[J]. PLoS One, 2018, 13(3):e0194628.
[35]	Wu JD, Jiang YL, Liang YN, et al. Expression of the maize MYB transcription factor ZmMYB3R enhances drought and salt stress tolerance in transgenic plants[J]. Plant Physiol Biochem, 2019, 137:179-188.
[36]	Xu ZJ, Sun ML, Jiang XF, et al. Glycinebetaine biosynthesis in response to osmotic stress depends on jasmonate signaling in watermelon suspension cells[J]. Front Plant Sci, 2018, 9:1469.
[37]	马超, 张均, 等. 外源MeJA对花后干旱胁迫下小麦光合特性的影响[J]. 麦类作物学报, 2018, 38(5):563-571.
	Ma C, Zhang J, et al. Effect of exogenous methyl jasmonate on photosynthetic characteristics in wheat under drought stress after anthesis[J]. J Triticeae Crops, 2018, 38(5):563-571.
[38]	Rouster J, Leah R, et al. Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain[J]. Plant J, 1997, 11(3):513-523.
[39]	Aquea F, Vega A, Timmermann T, et al. Genome-wide analysis of the set domain group family in grapevine[J]. Plant Cell Rep, 2011, 30(6):1087-1097.
[40]	Napsucialy-Mendivil S, Alvarez-Venegas R, Shishkova S, et al. Arabidopsis homolog of trithorax1(ATX1)is required for cell production, patterning, and morphogenesis in root development[J]. J Exp Bot, 2014, 65(22):6373-6384.
[41]	Choi SC, Lee S, Kim SR, et al. Trithorax group protein Oryza sativa trithorax1 controls flowering time in rice via interaction with early heading date3[J]. Plant Physiol, 2014, 164(3):1326-1337.
[42]	Hang RL, Liu CY, Ahmad A, et al. Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing[J]. PNAS, 2014, 111(45):16190-16195.
[43]	Han YF, et al. SUVR2 is involved in transcriptional gene silencing by associating with SNF2-related chromatin-remodeling proteins in Arabidopsis[J]. Cell Res, 2014, 24(12):1445-1465.
[44]	Luo YX, Han YF, Zhao QY, et al. Sumoylation of SUVR2 contributes to its role in transcriptional gene silencing[J]. Sci China Life Sci, 2018, 61(2):235-243.