基于转录组测序花烟草响应盐胁迫活性氧清除相关基因的挖掘

doi:10.13560/j.cnki.biotech.bull.1985.2020-0241

摘要/Abstract

摘要：

为了鉴定参与花烟草盐胁迫响应的活性氧清除相关基因,用200 mmol/L NaCl处理花烟草幼苗,并在处理后12 h采集其幼苗样本,提取总RNA,采用高通量测序技术,进行转录组测序。基于差异表达基因GO及KEGG分析的基础上,对参与其中的活性氧清除基因进行挖掘,利用qRT-PCR的方法验证转录组测序结果。结果表明,盐处理后,花烟草基因表达量变化在2倍以上的基因有7 239个（P<0.01）,其中,上调表达基因4 037个,下调表达基因3 162个,将其功能归类于生物过程、细胞组分及分子功能三大类159个GO条目,并显著富集在12条KEGG代谢通路中。同时,在盐胁迫下,花烟草有45个活性氧清除相关基因的表达发生了显著改变,其中,上调表达基因28个,下调表达基因17个。此结果说明,活性氧的清除是花烟草抵御盐胁迫的重要机制之一。

关键词: 花烟草, 转录组, 盐胁迫, 活性氧

Abstract:

In order to identify the genes related to reactive oxygen species（ROS）scavenging involved in salt stress response of Nicotiana tabacum seedlings,tobacco plants were treated with 200 mmol/L NaCl,and the samples of the seedlings were collected at 12 h after the treatment. After total RNA was extracted,high-throughput sequencing technology was carried out to sequence the transcriptome. On the basis of GO and KEGG analysis of the differentially expressed genes,ROS scavenging genes involved were mined,and the results of transcriptome sequencing were verified by qRT-PCR. The results showed that there were 7 239 genes（P < 0.01）with more than two-fold in the expression of tobacco genes after high salt treatment,among which 4 037 genes were up-regulated and 3 162 genes were down-regulated. The functions of these differentially expressed genes were classified into 159 GO items in three categories：biological process,cell components and molecular functions,and were significantly enriched in 12 KEGG metabolic pathways. Meanwhile,the expressions of 45 genes related to ROS scavenging processes changed significantly in tobacco plants under high salt stress,among which 28 genes were up-regulated and 17 genes were down-regulated. The results of this study suggest that the enhanced ROS scavenging ability could be one of the essential mechanisms of N. alata plants in response to salt stress.

Key words: Nicotiana alata, transcriptome, salt stress, reactive oxygen species（ROS）

鲁琳, 杨尚谕, 刘维东, 鲁黎明. 基于转录组测序花烟草响应盐胁迫活性氧清除相关基因的挖掘[J]. 生物技术通报, 2020, 36(12): 42-53.

LU Lin, YANG Shang-yu, LIU Wei-dong, LU Li-ming. Mining of Genes Related to Reactive Oxygen Species Scavenging in Response to Salt Stress in Nicotiana alata Based on Transcriptome Sequencing[J]. Biotechnology Bulletin, 2020, 36(12): 42-53.

图/表 9

参考文献 42

[1]	Soares ALC, Geilfus CM, Carpentier SC. Genotype-specific growth and proteomic responses of maize toward salt stress[J]. Frontier in Plant Science, 2018,9:661.
[2]	Acosta-Motos JR, Ortuño MF, Bernal-Vicente A, et al. Plant responses to salt stress:adaptive mechanisms[J]. Agronomy, 2017,7:1-38. doi: 10.3390/agronomy7010001 URL
[3]	Yang Y, Guo Y. Elucidating the molecular mechanisms mediating plant salt-stress responses[J]. New Phytologist, 2018,217:523-539.
[4]	Noctor G, Reichheld JP, Foyer CH. ROS-related redox regulation and signaling in plants[J]. Seminars in Cell & Developmental Biology, 2018,80:3-12.
[5]	马静, 王静, 姚鹏伟, 等. 盐分胁迫对烤烟叶片亚细胞结构及生理生化指标的影响[J]. 中国烟草学报, 2019,25(5):44-52.
	Ma J, Wang J, Yao PW, et al. Effects of salt stress on subcellular structure and physiological and biochemical indexes of flue-cured tobacco leaves[J]. Acta Tabacaria Sinica, 2019,25(5):44-52.
[6]	李猛, 陈栋, 李秀妮, 等. 盐胁迫下外源褪黑素对烟草幼苗抗氧化特性和光合特性的影响[J]. 中国农业科技导报, 2019,21(2):141-147.
	Li M, Chen D, Li XN, et al. Influenes of exogenous melatonin on antioxidant and photosynthetic characteristics of tobacco seedlings under salt stress[J]. Journal of Agricultural Science and Technology, 2019,21(2):141-147.
[7]	Singh N, Mishra A, Jha B. Over-expression of the peroxisomal ascorbate peroxidase(SbpAPX)gene cloned from halophyte Salicornia brachiate confers salt and drought stress tolerance in transgenic tobacco[J]. Marine Biotechnology, 2014,16(3):321-332. URL pmid: 24197564
[8]	Badawi GH, Yamauchi Y, Shimada E, et al. Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco(Nicotiana tabacum)chloroplasts[J]. Plant Science, 2004,166:919-928.
[9]	Prashanth SR, Sadhasivam V, Parida A. Overexpression of cytosolic copper/zinc superoxide dismutase from a mangrove plant Avicennia marina in indica rice var Pusa Basmati-1 confers abiotic stress tolerance[J]. Transgenic Research, 2008,17:281-291. URL pmid: 17541718
[10]	Wang Y, Ying Y, Chen J, et al. Transgenic Arabidopsis overexpressing Mn-SOD enhanced salt-tolerance[J]. Plant Science, 2004,167:671-677.
[11]	Wang YC, Qu GZ, Li HY, et al. Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from Tamarix androssowii[J]. Molecular Biology Report, 2010,37:1119-1124.
[12]	Kim YH, Kim CY, Song WK, et al. Overexpression of sweet potato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco[J]. Planta, 2008,227(4):867-881. doi: 10.1007/s00425-007-0663-3 URL pmid: 18224366
[13]	Al-Taweel K, Iwaki T, Yabuta Y, et al. A bacterial transgene for catalase protects translation of d1 protein during exposure of salt-stressed tobacco leaves to strong light[J]. Plant Physiology, 2007,145(1):258-265. doi: 10.1104/pp.107.101733 URL pmid: 17660354
[14]	Xu WF, Shi WM, Ueda A, et al. Mechanisms of salt tolerance in transgenic Arabidopsis thaliana carrying a peroxisomal ascorbate peroxidase gene from barley[J]. Pedosphere, 2008,18:486-495.
[15]	Diaz-Vivancos P, Faize M, Barba-Espin G, et al. Ectopic expression of cytosolic superoxide dismutase and ascorbate per-oxidase leads to salt stress tolerance in transgenic plums[J]. Plant Biotechnology, 2013,11:976-985.
[16]	Gaber A, Yoshimura K, Yamamoto T, et al. Glutathione peroxidase-like protein of Synechocystis PCC 6803 confers tolerance to oxidative and environmental stresses in transgenic Arabidopsis[J]. Physiology Plantarum, 2006,128:251-262.
[17]	Yoshimura K, Miyao K, Gaber A, et al. Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol[J]. Plant Journal, 2004,37:21-33.
[18]	Luo Q, Teng W, Fang S, et al. Transcriptome analysis of salt-stress response in three seedling tissues of common wheat[J]. The Crop Journal, 2019,3:378-392.
[19]	Zenda T, Liu S, Wang X, et al. Key maize drought-responsive genes and pathways revealed by comparative transcriptome and physiological analyses of contrasting inbred lines[J]. International Journal of Molecular Sciences, 2019,20, 1268.
[20]	朱琳, 袁梦, 高红秀, 等. 水稻苗期低温应答转录组分析[J]. 华北农学报, 2018,33(5):40-51.
	Zhu L, Yuan M, Gao H, et al. Transcriptomic analysis of rice seedling responsive to low temperature[J]. Acta Agriculturae Boreali-Sinica, 2018,33(5):40-51.
[21]	鲁黎明, 陈勇, 鲁逸飞, 等. 低钾胁迫对烟草幼苗叶中基因表达的影响[J]. 核农学报, 2016,30(7):1273-1280.
	Lu LM, Chen Y, Lu YF, et al. Effect of low potassium stress on gene expression profile in leaves of tobacco seedlings[J]. Journal of Nuclear Agricultural Sciences, 2016,30(7):1273-1280.
[22]	Shigeoka S, Ishikawa T, Tamoi M, et al. Regulation and function of ascorbate peroxidase isoenzymes[J]. Jounal of Experimental Botany, 2002,53(372):1305-1319.
[23]	马长乐, 王萍萍, 曹子谊, 等. 盐地碱蓬(Suaede sdlsa)APX 基因的克隆及盐胁迫下的表达[J]. 植物生理与分子生物学学报, 2002,28(4):261-266.
	Ma CL, Wang PP, Cao ZY, et al. cDNA cloning and gene expression of APX in Suaeda salsa in response to salt stress[J]. Journal of Plant Physiology and Molecular Biology, 2002,28(4):261-266.
[24]	王超, 杨传平, 王玉成. 白桦抗坏血酸过氧化物酶(APX)基因克隆及表达分析[J]. 东北林业大学学报, 2009,37(3):70-81.
	Wang C, Yang CP, Wang YC, et al. Cloning and expression analysis of an APX gene from Betula platyphylla[J]. Journal of Northeast Forestry University, 2009,37(3):70-81.
[25]	Badawi GH, Kawano N, Yamauchi Y, et al. Overexpression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit[J]. Physiology Plantarum, 2004,121(2):231-238.
[26]	郭慧娜, 孟玉平, 郝子琪, 等. 转ZjAPX 基因拟南芥对NaCl、干旱胁迫的耐性研究[J]. 生物技术通报, 2013,29(1):78-82.
	Guo HN, Meng YP, Hao ZQ, et al. ZjAPX gene improving resistance to NaCl and drought stress in Arabidopsis[J]. Biotechnology Bulletin, 2013,29(1):78-82.
[27]	乔新荣, 张继英. 植物谷胱甘肽过氧化物酶(GPX)研究进展 [J]. 生物技术通报, 2016,32(9):7-13.
	Qiao XR, Zhang JY. Research progress on GPX in plants[J]. Biotechnology Bulletin, 2016,32(9):7-13.
[28]	Yoshimura K, Miyao K, Gaber A, et al. Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydom-onas glutathione peroxidase in cytosol or chloroplast[J]. Plant Journal, 2004,37(1):21-33.
[29]	Zhai CZ, Zhao L, Yin LJ, et al. Two wheat glutathione peroxidase genes whose products are located in chloroplasts improve salt and H₂O₂ tolerances in Arabidopsis[J]. PLoS One, 2013,8(10):e73989. URL pmid: 24098330
[30]	Akbudak MA, Filiz E, Vatansever R, et al. Genome wide identification and expression profile of ascorbate peroxidase(APX)and glutathione peroxidase(GPX)genes under drought stress in Sorghum(Sorghum bicolor L.)[J]. Journal of Plant Growth Regulation, 2018,37(3):925-936.
[31]	Labrou NE, Papageorgiou AC, Pavli O, et al. Plant GSTome:Structure and functional role in xenome network and plant stress response[J]. Current Opinion of Biotechnology, 2015,32:186-194. doi: 10.1016/j.copbio.2014.12.024 URL
[32]	陈秀华, 王臻昱, 李先平, 等. 谷胱甘肽S-转移酶的研究进展[J]. 东北农业大学学报, 2013,44(1):149-153.
	Chen XH, Wang ZY, Li XP, et al. Research progress on glutathione S-transferases[J]. Journal of Northeast Agricultural University, 2013,44(1):149-153.
[33]	Xu J, Tian YS, Xing XJ, et al. Over-expression of AtGSTU19 provides tolerance to salt, drought and methy viologen stresses in Arabidopsis[J]. Physiology Plantarum, 2015,156(2):164-175. doi: 10.1111/ppl.2016.156.issue-2 URL
[34]	Roxas VP, Smith RK Jr, Allen ER, et al. Overexpression of glutathione S-transferase glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress[J]. Nature Biotechnology, 1997,15:988-991. URL pmid: 9335051
[35]	戚元成, 张世敏, 王丽萍, 等. 谷胱甘肽转移酶基因过量表达能加速盐胁迫下转基因拟南芥的生长[J]. 植物生理与分子生物学学报, 2004,30(5):517-522. pmid: 15627705
	Qi YC, Zhang SM, Wang LP, et al. Overexpression of GST gene accelerates the growth of transgenic Arabidopsis under salt stress[J]. Journal of Plant Physiology and Molecular Biology, 2004,30(5):517-522. URL pmid: 15627705
[36]	Chen JH, Jiang HW, Hsieh EJ, et al. Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid[J]. Plant Physiology, 2012,158(1):340-351. doi: 10.1104/pp.111.181875 URL pmid: 22095046
[37]	Gill SS, Anjum NA, Hasanuzzaman M, et al. Glutathione and glutathione reductase:A boon in disguise for plant abiotic stress defense operations[J]. Plant Physiology and Biochemistry, 2013,70:204-212. doi: 10.1016/j.plaphy.2013.05.032 URL pmid: 23792825
[38]	Wu T, Lin W, Kao Y, et al. Identification and characterization of a novel chloroplast /mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice[J]. Plant Molecular Biology, 2013,83:379-390. doi: 10.1007/s11103-013-0095-3 URL pmid: 23783412
[39]	岳川, 曹红利, 周艳华, 等. 茶树谷胱甘肽还原酶基因 CsGRs 的克隆与表达分析[J]. 中国农业科学, 2014,47(16):3277-3289. doi: 10.3864/j.issn.0578-1752.2014.16.013 URL
	Yue C, Cao HL, Zhou YH, et al. Cloning and expression analysis of glutathione reductase genes(CsGRs) in tea plant(Camellia sinensis)[J]. Scientia Agricultura Sinica, 2014,47(16):3277-3289. doi: 10.3864/j.issn.0578-1752.2014.16.013 URL
[40]	张腾国, 聂亭亭, 孙万仓, 等. 逆境胁迫对油菜谷胱甘肽还原酶基因表达及其酶活性的影响[J]. 应用生态学报, 2018,29(1):213-222. pmid: 29692030
	Zhang TG, Nie TT, Sun WC, et al. Effects of diverse stresses on gene expression and enzyme activity of glutathione reductase in Brassica campestris[J]. Chinese Journal of Applied Ecology, 2018,29(1):213-222. doi: 10.13287/j.1001-9332.201801.010 URL pmid: 29692030
[41]	Deng Z, Zhao M, Liu H, et al. Molecular cloning, expression profiles and characterization of a glutathione reductase in Hevea brasiliensis[J]. Plant Physiology and Biochemistry, 2015,96:53-63. doi: 10.1016/j.plaphy.2015.07.022 URL pmid: 26232647
[42]	Wu TM, Lin WR, Kao CH, et al. Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice[J]. Plant Molecular Biology, 2015,87:555-564. doi: 10.1007/s11103-015-0290-5 URL pmid: 25636203

序号	引物名称	引物序列（5'-3'）
1	GST1-F	CCGGCAATAAGCATGAAGAT
1	GST1-R	TAGTATCCCCTTGGGTGTGG
2	CAT1-F	CCAATAAGCTACCACTACGAAACG
2	CAT1-R	GGGAAAGAGGAGTGAACCCATT
3	POD42-F	AAGAGCCATCACTATCTGGTACAAAG
3	POD42-R	CTGCCTCTCCGTCTGAACCT
4	CIPK11-F	GCGTTGACGGAGCAAGTACA
4	CIPK11-R	ATGCTGGCGTTCCACAAAG
5	ERF4-F	TCACTACGACTGGGCTTACCAA
5	ERF4-R	GGAATTGCTCAATGTTTGTCCTAA
6	GPX4-F	CCCTGATGTTCCACATGAGATCT
6	GPX4-R	GGGTGGAAAGGAAACACAGTACA
7	MYB5-F	GTGAAGGCCAATGGAGAAACTT
7	MYB5-R	TCTTAGCCTACAACTTTTTCCACATC
8	CIPK6-F	TCCGCCTTGGTTTTCATCAG
8	CIPK6-R	TCGGATTCGGATCCAACATT
9	Actin-F	CCAAGCAGCATGAAGATCAA
9	Actin-R	CGTACTCGGCCTTTGAAATC

序号	引物名称	引物序列（5'-3'）
1	GST1-F	CCGGCAATAAGCATGAAGAT
1	GST1-R	TAGTATCCCCTTGGGTGTGG
2	CAT1-F	CCAATAAGCTACCACTACGAAACG
2	CAT1-R	GGGAAAGAGGAGTGAACCCATT
3	POD42-F	AAGAGCCATCACTATCTGGTACAAAG
3	POD42-R	CTGCCTCTCCGTCTGAACCT
4	CIPK11-F	GCGTTGACGGAGCAAGTACA
4	CIPK11-R	ATGCTGGCGTTCCACAAAG
5	ERF4-F	TCACTACGACTGGGCTTACCAA
5	ERF4-R	GGAATTGCTCAATGTTTGTCCTAA
6	GPX4-F	CCCTGATGTTCCACATGAGATCT
6	GPX4-R	GGGTGGAAAGGAAACACAGTACA
7	MYB5-F	GTGAAGGCCAATGGAGAAACTT
7	MYB5-R	TCTTAGCCTACAACTTTTTCCACATC
8	CIPK6-F	TCCGCCTTGGTTTTCATCAG
8	CIPK6-R	TCGGATTCGGATCCAACATT
9	Actin-F	CCAAGCAGCATGAAGATCAA
9	Actin-R	CGTACTCGGCCTTTGAAATC

样品名称	原始数据	筛选后数据	总映射	多重映射	唯一映射	Q30/%	GC含量/%
CK_1	49583710	47599094	44085400（92.62%）	969397（2.04%）	43116003（90.58%）	90.32	42.98
CK_2	56621858	54483002	50100087（91.96%）	1130193（2.07%）	48969894（89.88%）	90.99	43.03
CK_3	50230428	47972778	44659130（93.09%）	965148（2.01%）	43693982（91.08%）	90.53	43.03
S12_1	64969188	62591696	57912308（92.52%）	1294796（2.07%）	56617512（90.46%）	90.33	43.00
S12_2	52595884	50762784	47212736（93.01%）	1022342（2.01%）	46190394（90.99%）	90.38	42.97
S12_3	53054528	51241836	47521067（92.74%）	1099309（2.15%）	46421758（90.59%）	90.57	43.19

样品名称	原始数据	筛选后数据	总映射	多重映射	唯一映射	Q30/%	GC含量/%
CK_1	49583710	47599094	44085400（92.62%）	969397（2.04%）	43116003（90.58%）	90.32	42.98
CK_2	56621858	54483002	50100087（91.96%）	1130193（2.07%）	48969894（89.88%）	90.99	43.03
CK_3	50230428	47972778	44659130（93.09%）	965148（2.01%）	43693982（91.08%）	90.53	43.03
S12_1	64969188	62591696	57912308（92.52%）	1294796（2.07%）	56617512（90.46%）	90.33	43.00
S12_2	52595884	50762784	47212736（93.01%）	1022342（2.01%）	46190394（90.99%）	90.38	42.97
S12_3	53054528	51241836	47521067（92.74%）	1099309（2.15%）	46421758（90.59%）	90.57	43.19

GO分类号	功能描述	差异基因数量（DEGs）	差异基因占比 /%
GO：0008150	Biological_process	4 595	76.97
GO：0008152	Metabolic process	3 660	61.31
GO：0044699	Single-organism process	2 808	47.04
GO：0044763	Single-organism cellular process	2 047	34.29
GO：0009058	Biosynthetic process	1 795	30.07
GO：0044710	Single-organism metabolic process	1 730	28.98
GO：0051234	Establishment of localization	1 121	18.78
GO：0006810	Transport	1 119	18.74
GO：1902578	Single-organism localization	845	14.15
GO：0044765	Single-organism transport	836	14.00
GO：0055114	Oxidation-reduction process	788	13.20
GO：0006796	Phosphate-containing compound metabolic process	693	11.61
GO：0006793	Phosphorus metabolic process	693	11.61
GO：0044711	Single-organism biosynthetic process	646	10.82
GO：0006464	Cellular protein modification process	563	9.43
GO：0036211	Protein modification process	563	9.43
GO：0005975	Carbohydrate metabolic process	549	9.20
GO：0044281	Small molecule metabolic process	535	8.96
GO：0006629	Lipid metabolic process	524	8.78
GO：0055085	Transmembrane transport	464	7.77
GO：0006811	Ion transport	460	7.71
GO：0016310	Phosphorylation	449	7.52
GO：1901135	Carbohydrate derivative metabolic process	428	7.17
GO：0044255	Cellular lipid metabolic process	366	6.13
GO：0006812	Cation transport	361	6.05
GO：0044723	Single-organism carbohydrate metabolic process	359	6.01
GO：0008610	Lipid biosynthetic process	320	5.36
GO：0019637	Organophosphate metabolic process	309	5.18
GO：1901137	Carbohydrate derivative biosynthetic process	267	4.47
GO：0044262	Cellular carbohydrate metabolic process	240	4.02