基于转录组测序的花烟草低温胁迫响应转录因子挖掘

doi:10.7525/j.issn.1673-5102.2021.01.015

摘要/Abstract

摘要：

为了鉴定参与花烟草低温胁迫的转录因子，对花烟草幼苗进行了4℃低温处理，并在处理后12 h采集其幼苗样本，提取总RNA后，采用高通量测序技术，进行了转录组测序。在对差异表达基因进行GO及KEGG分析的基础上，对参与其中的转录因子进行了挖掘，并采用qRT-PCR的方法，对转录组测序的结果进行了验证。结果表明，低温处理后，花烟草基因表达量变化在2倍以上的基因有8 388个（P<0.01），其中，上调表达4 229个，下调表达4 159个。这些差异表达基因的功能归类于生物过程、细胞组分及分子功能3大类69个GO条目，并显著富集在40条KEGG代谢通路中。同时，在低温胁迫下，花烟草有118个转录因子的表达发生了显著改变，其中，上调表达82个，下调表达36个。这些转录因子属于28个家族，其中，数量最多的为NAC家族19个，其次为ERF家族16个、MYB家族15个、WRKY家族15个。本研究的结果为花烟草低温响应分子机制的研究提供了借鉴。

关键词: 花烟草, 转录组, 低温胁迫, 转录因子

Abstract:

In order to identify the transcription factors involved in the low temperature stress of Nicotiana alata seedlings， 4℃ low temperature was carried out， and samples of the seedlings were collected in 12-hour treatment， then the total RNA was extracted， and high-throughput sequencing technology was used to sequence the transcriptome. For differentially expressed genes， GO and KEGG analysis were applied， the transcription factors involved were classified， and the results of transcriptome sequencing were verified by qRT-PCR. There were 8 388 genes（P<0.01） whose expression changed more than twice after low temperature treatment， of which 4 229 genes were up-regulated and 4 159 genes were down regulated. The functions of these differentially expressed genes were classified into three categories： biological process， cell components and molecular functions， 69 items in go entries， and were significantly enriched in 40 KEGG metabolic pathways. At the same time， under low temperature stress， the expression of 118 transcription factors in N.alata changed significantly， among which 82 genes were up-regulated and 36 genes were down regulated. These transcription factors belonged to 28 families， of which 19 genes to NAC family， 16 genes to ERF family， 15 genes to MYB family and 15 genes to WRKY family. The results provided a reference for the study of the molecular mechanism of low temperature response in N.alata.

Key words: Nicotiana alata, transcriptome, low temperature stress, transcription factors

中图分类号:

S572

鲁琳, 杨尚谕, 刘维东, 鲁黎明. 基于转录组测序的花烟草低温胁迫响应转录因子挖掘[J]. 植物研究, 2021, 41(1): 119-129.

Lin LU, Shang-Yu YANG, Wei-Dong LIU, Li-Ming LU. To Explore the Transcription Factor in Response to Low Temperature Stress in Nicotiana alata by Transcriptome Sequencing[J]. Bulletin of Botanical Research, 2021, 41(1): 119-129.

图/表 8

表1

表2

图1

表3

上调差异表达基因的GO功能注释分析

功能描述 Description	分类 Term_type	差异基因数量 DEG_item
Cellular carbohydrate metabolic process	Biological process	256
Cellular carbohydrate biosynthetic process		212
Single-organism carbohydrate metabolic process		371
Carbohydrate biosynthetic process		232
Cellular polysaccharide metabolic process		205
Phosphorylation		521
Cellular polysaccharide biosynthetic process		184
Polysaccharide metabolic process		222
Phosphate-containing compound metabolic process		761
Polysaccharide biosynthetic process		185
Photosynthetic electron transport chain		19
Lipopolysaccharide metabolic process		128
Lipopolysaccharide biosynthetic process		128
Phosphorus metabolic process		761
Protein phosphorylation		447
Cellular lipid metabolic process		337
Lipid biosynthetic process		292
Lipid metabolic process		471
Coenzyme metabolic process		147
Carbohydrate metabolic process		537
Cellular protein modification process		634
Protein modification process		634
Carbohydrate catabolic process		77
Oxidoreduction coenzyme metabolic process		84
Nucleoside diphosphate phosphorylation		49
Nucleotide phosphorylation		49
Nucleoside diphosphate metabolic process		49
Pyridine-containing compound metabolic process		78
Pyruvate metabolic process		44
Glycolytic process		43
ATP generation from ADP		43
Purine nucleoside diphosphate metabolic process		43
Purine ribonucleoside diphosphate metabolic process		43
Ribonucleoside diphosphate metabolic process		43
ADP metabolic process		43
Pyridine nucleotide metabolic process		74
Nicotinamide nucleotide metabolic process		74
Liposaccharide metabolic process		167
Response to hormone		37
Single-organism carbohydrate catabolic process		56
Cellulose metabolic process		33
Macromolecule modification		683
Carbohydrate derivative biosynthetic process		287
Photosynthetic electron transport in photosystem II		9
Response to endogenous stimulus		38
Inositol metabolic process		9
Cofactor metabolic process		229
Cellulose biosynthetic process		29
Carbohydrate derivative metabolic process		409
beta-glucan metabolic process		39
Inositol biosynthetic process		6
Polyol biosynthetic process		6
beta-glucan biosynthetic process		35
Protein kinase activity	Molecular function	503
Kinase activity		638
Phosphotransferase activity, alcohol group as acceptor		599
Chlorophyll binding		13
Inorganic phosphate transmembrane transporter activity		8
Fructose-bisphosphate aldolase activity		13
Intramolecular lyase activity		13
Transferase activity, transferring phosphorus-containing groups		948
Transferase activity		1 558
Inositol-3-phosphate synthase activity		6

表3

表4

图2

图3

图4

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序号 ID	引物名称 Primers	引物序列 Sequence(5′—3′)
1	IAA17-F	TGTTGGAGATGTTCCTTGGAAA
1	IAA17-R	GGCTTCCGAGCTCTTCATCA
2	NAC2-F	CCCAGAGGAATCAAAACCAA
2	NAC2-R	CCAATCGTCAAGCCTCAAGTTA
3	P450-F	TGGCTTTGGTGACGCTAAAT
3	P450-R	GCTGCCAGTCCTGTATTTCC
4	WRKY6-F	TGTCCCCGAGCTTATTATCG
4	WRKY6-R	TGGTAGGGGGTGGTTATGTG
5	CIPK6-F	AAGCTATGGATTCATCTGTTACATTG
5	CIPK6-R	TTTCATTTCAAGAGACATACCTCAA
6	ERF025-F	CAGATTGGCTAGAAGCACCA
6	ERF025-R	ACAAACTGGGAGCCACTACC
7	Actin-F	CCAAGCAGCATGAAGATCAA
7	Actin-R	CGTACTCGGCCTTTGAAATC

Sample name	Raw reads	Clean reads	Total mapped	Multiple mapped	Uniquely mapped	Q30(%)	GC content(%)
CK_1	49 583 710	47 599 094	4 4085 400(92.62%)	969 397(2.04%)	43 116 003(90.58%)	90.32	42.98
CK_2	56 621 858	54 483 002	5 0100 087(91.96%)	1 130 193(2.07%)	48 969 894(89.88%)	90.99	43.03
CK_3	50 230 428	47 972 778	4 4659 130(93.09%)	965 148(2.01%)	43 693 982(91.08%)	90.53	43.03
F12_1	59 737 702	57 148 708	4 7984 291(83.96%)	1 208 247(2.11%)	46 776 044(81.85%)	89.15	39.37
F12_2	60 120 416	57 845 042	4 9904 446(86.27%)	1 231 084(2.13%)	48 673 362(84.14%)	90.09	40.06
F12_3	50 761 464	48 721 146	4 2563 180(87.36%)	1 011 326(2.08%)	41 551 854(85.29%)	89.78	40.68

功能描述 Description	分类 Term_type	差异基因数量 DEG_item
Nuclear ubiquitin ligase complex	Cellular component	96
Anaphase-promoting complex		96
Cullin-RING ubiquitin ligase complex		101
Ubiquitin ligase complex		125
Ubiquitin-protein transferase activity	Molecular function	128
Ubiquitin-like protein transferase activity	Molecular function	128

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