中国药学杂志

Cultivars name	Tissue site	Sample number(n=3)
Mingui 1(M1)	Leaf	M1L-1、M1L-2、M1L-3
	Root	M1R-1、M1R-2、M1R-3
Mingui 2(M2)	Leaf	M2L-1、M2L-2、M2L-3
	Root	M2R-1、M2R-2、M2R-3

Cultivars name	Tissue site	Sample number(n=3)
Mingui 1(M1)	Leaf	M1L-1、M1L-2、M1L-3
	Root	M1R-1、M1R-2、M1R-3
Mingui 2(M2)	Leaf	M2L-1、M2L-2、M2L-3
	Root	M2R-1、M2R-2、M2R-3

Gene name	Primer sequence (5-3)	Amplification length/bp
HVA22C	F:TTCAACGGAGCTAGTATTGCCTACG	175
	R:TCTCCAAAGCCTCAGTTCCATTCTC
KRP1	F:GGTGATGTGAATGGAGATGGAGTTC	106
	R:CTTCTTCAAGCCAAACCTCAGATTC
PUB23	F:ACGGCATCCAAAGAATCCCAACTC	175
	R:TAACCCGCTCCTGCTGCTTCC
DREB1B	F:GTGTTGCCAGCTTCCAGTAATCC	93
	R:TCTACGCCTCACTCCTCTGTAAAC
Bp10	F:TCCAAGACCCAACCCACAAGG	147
	R:AGAGGTGTATCCGATGGTATGAAGG
ACT	F:TGGTATTGTGCTGGATTCTGGT	109
	R:TGAGATCACCACCAGCAAGG

Gene name	Primer sequence (5-3)	Amplification length/bp
HVA22C	F:TTCAACGGAGCTAGTATTGCCTACG	175
	R:TCTCCAAAGCCTCAGTTCCATTCTC
KRP1	F:GGTGATGTGAATGGAGATGGAGTTC	106
	R:CTTCTTCAAGCCAAACCTCAGATTC
PUB23	F:ACGGCATCCAAAGAATCCCAACTC	175
	R:TAACCCGCTCCTGCTGCTTCC
DREB1B	F:GTGTTGCCAGCTTCCAGTAATCC	93
	R:TCTACGCCTCACTCCTCTGTAAAC
Bp10	F:TCCAAGACCCAACCCACAAGG	147
	R:AGAGGTGTATCCGATGGTATGAAGG
ACT	F:TGGTATTGTGCTGGATTCTGGT	109
	R:TGAGATCACCACCAGCAAGG

No.	Raw reads	Clean reads/%	Clean reads/bp	Q20/%	Q30/%	GC/%	Mapped reads	Mapped ratio/%
M1L-1	45 403 718	45 123 184(99.38)	6 673 603 863	97.97	93.75	42.83	40 424 439	89.59
M1L-2	56 479 970	56 136 434(99.39)	8 310 128 766	97.79	93.39	42.95	50 542 876	90.04
M1L-3	51 450 794	51 103 374(99.32)	7 610 806 624	97.64	93.00	42.91	45 316 441	88.68
M2L-1	49 972 658	49 665 860(99.39)	7 374 089 668	97.87	93.53	43.14	44 735 242	90.07
M2L-2	47 873 956	47 583 270(99.39)	7 024 011 837	97.91	93.66	43.10	41 434 239	87.08
M2L-3	49 948 026	49 632 084(99.37)	7 375 539 235	97.47	92.64	42.93	44 482 152	89.62
M1R-1	46 193 770	45 851 916(99.26)	6 790 909 357	97.60	92.96	43.15	37 046 874	80.80
M1R-2	48 730 572	48 436 458(99.40)	7 068 058 117	97.95	93.82	43.04	38 902 366	80.32
M1R-3	46 580 816	46 266 062(99.32)	6 862 726 173	97.69	93.13	42.91	37 217 870	80.44
M2R-1	43 229 068	42 923 476(99.29)	6 341 975 820	97.58	92.88	43.01	34 686 284	80.81
M2R-2	55 754 104	55 400 420(99.37)	8 232 259 847	97.58	92.90	42.78	42 144 671	76.07
M2R-3	46 570 526	46 300 698(99.42%)	6 884 153 864	97.69	93.10	42.95	36 909 107	79.72

No.	Raw reads	Clean reads/%	Clean reads/bp	Q20/%	Q30/%	GC/%	Mapped reads	Mapped ratio/%
M1L-1	45 403 718	45 123 184(99.38)	6 673 603 863	97.97	93.75	42.83	40 424 439	89.59
M1L-2	56 479 970	56 136 434(99.39)	8 310 128 766	97.79	93.39	42.95	50 542 876	90.04
M1L-3	51 450 794	51 103 374(99.32)	7 610 806 624	97.64	93.00	42.91	45 316 441	88.68
M2L-1	49 972 658	49 665 860(99.39)	7 374 089 668	97.87	93.53	43.14	44 735 242	90.07
M2L-2	47 873 956	47 583 270(99.39)	7 024 011 837	97.91	93.66	43.10	41 434 239	87.08
M2L-3	49 948 026	49 632 084(99.37)	7 375 539 235	97.47	92.64	42.93	44 482 152	89.62
M1R-1	46 193 770	45 851 916(99.26)	6 790 909 357	97.60	92.96	43.15	37 046 874	80.80
M1R-2	48 730 572	48 436 458(99.40)	7 068 058 117	97.95	93.82	43.04	38 902 366	80.32
M1R-3	46 580 816	46 266 062(99.32)	6 862 726 173	97.69	93.13	42.91	37 217 870	80.44
M2R-1	43 229 068	42 923 476(99.29)	6 341 975 820	97.58	92.88	43.01	34 686 284	80.81
M2R-2	55 754 104	55 400 420(99.37)	8 232 259 847	97.58	92.90	42.78	42 144 671	76.07
M2R-3	46 570 526	46 300 698(99.42%)	6 884 153 864	97.69	93.10	42.95	36 909 107	79.72

Sort	Subclassification	Gene abbreviation	Comment name	log₂FC	Expression pattern
Abscisic acid regulation		DREB1B	Dehydration-responsive element-binding protein 1B	2.96	3.03	UR
		DREB1D	Dehydration-responsive element-binding protein 1D	2.82	2.30	UR
		ERD7	Protein early-responsive to dehydration 7	1.58	1.18	UR
		ERD15	Protein early-responsive to dehydration 15	1.54	1.41	UR
		HVA22A	HVA22-like protein a	1.82	1.84	UR
		HVA22C	HVA22-like protein c	1.94	2.87	UR
		MPK3	Mitogen-activated protein kinase 3	2.16	2.02	UR
		FER	Receptor-like protein kinase FERONIA	1.83	1.11	UR
		PUB23	E3 ubiquitin-protein ligase PUB23	1.99	2.49	UR
		PUB9	U-box domain-containing protein 9	1.85	1.20	UR
Osmotic regulation		AGP41	Arabinogalactan protein 41	1.46	1.48	UR
		APF2	Aspartyl protease family protein 2	1.10	2.05	UR
		ASP1	Aspartic proteinase Asp1	1.82	1.65	UR
		At3g56050	Probable inactive receptor-like protein kinase At3g56050	1.28	1.29	UR
		At4g16563	Probable aspartyl protease At4g16563	1.63	1.54	UR
		ERD4	CSC1-like protein ERD4	1.31	1.35	UR
		NTL9	Protein NTM1-like 9 (Calmodulin-binding NAC protein)	1.15	1.37	UR
		PERK1	Proline-rich receptor-like protein kinase PERK1	1.70	2.64	UR
		KRP1	Calcium-binding protein KRP1	3.68	1.78	UR
		RALFL33	Protein RALF-like 33	1.56	1.17	UR
		RXW8	CSC1-like protein RXW8	1.43	2.03	UR
		TPPJ	Probable trehalose-phosphate phosphatase J	2.59	1.16	UR
		TPS7	Probable a-trehalose-phosphate synthase	1.16	1.23	UR
Reactive oxygen scavenging		AAO	L-ascorbate oxidase (ASO)	1.62	2.33	UR
		AO	L-aspartate oxidase	1.04	1.21	UR
		At3g08610	NADH dehydrogenase [ubiquinone]	-1.98	-2.36	DR
		Bp10	L-ascorbate oxidase homolog	3.55	4.50	UR
		CYP76A2	Cytochrome P450 76A2	1.29	2.51	UR
		SDR2a	Short-chain dehydrogenase reductase 2a	2.15	2.08	UR
Regulation of other	Aquaporin	SYP121	Protein Syntaxin of plants 121	2.70	1.89	UR
functional proteins	Response protein	At2g40140	Zinc finger CCCH domain-containing protein 29	1.78	1.01	UR
		At4g27290	G-type lectin S-receptor-like serine/threonine-protein kinase At4g27290	2.32	1.25	UR
		ATL6	E3 ubiquitin-protein ligase ATL6	1.18	1.46	UR
		BAP2	BON1-associated protein 2	2.30	1.54	UR
		DGK1	Diacylglycerol kinase 1	1.01	1.49	UR
		DGK2	Diacylglycerol kinase 2	2.64	2.19	UR
		FLS2	LRR receptor-like serine/threonine-protein kinase FLS2	1.41	2.93	UR
		GATA8	GATA transcription factor 8	2.80	1.17	UR
		NCL	Sodium/calcium exchanger NCL	2.59	2.69	UR
		NHL10	NDR1/HIN1-like protein 10	1.55	1.36	UR
		NHL13	NDR1/HIN1-like protein 13	2.32	1.02	UR
		PBL3	Probable serine/threonine-protein kinase PBL3	1.56	1.51	UR
		PCR2	Protein plant cadmium resistance 2	1.56	1.58	UR
		PCRK1	Serine/threonine-protein kinase PCRK1	1.24	1.01	UR
		RPV1	Disease resistance protein RPV1	2.22	2.17	UR
		WRKY40	Probable WRKY transcription factor 40	1.58	2.49	UR
		WRKY53	Probable WRKY transcription factor 53	1.13	1.53	UR
	Cell wall construction	CDC20-1	Cell division cycle 20.1, cofactor of APC complex	1.92	1.23	UR
		CESA2	Cellulose synthase A catalytic subunit 2	3.21	2.21	UR
		CHIT3	Hevamine-A	1.48	1.13	UR
		CSLC12	Probable xyloglucan glycosyltransferase 12	1.14	1.27	UR
		DCR	BAHD acyltransferase DCR	1.65	1.82	UR
		MUCI21	Xylan glycosyltransferase MUCI21	1.66	1.51	UR
		TBR	Protein trichome birefringence	1.39	1.07	UR
		THE1	Receptor-like protein kinase THESEUS 1	1.67	1.08	UR
		XTH15	Xyloglucan endotransglucosylase/hydrolase 15	2.59	1.73	UR
		XTH22	Xyloglucan endotransglucosylase/hydrolase protein 22	2.85	1.27	UR
		XTH23	Probable xyloglucan endotransglucosylase/hydrolase protein 23	3.07	2.35	UR
		XXT3	Probable xyloglucan 6-xylosyltransferase 3	1.60	1.66	UR
		XXT5	Probable xyloglucan 6-xylosyltransferase 5	1.63	1.26	UR

Sort	Subclassification	Gene abbreviation	Comment name	log₂FC	Expression pattern
Abscisic acid regulation		DREB1B	Dehydration-responsive element-binding protein 1B	2.96	3.03	UR
		DREB1D	Dehydration-responsive element-binding protein 1D	2.82	2.30	UR
		ERD7	Protein early-responsive to dehydration 7	1.58	1.18	UR
		ERD15	Protein early-responsive to dehydration 15	1.54	1.41	UR
		HVA22A	HVA22-like protein a	1.82	1.84	UR
		HVA22C	HVA22-like protein c	1.94	2.87	UR
		MPK3	Mitogen-activated protein kinase 3	2.16	2.02	UR
		FER	Receptor-like protein kinase FERONIA	1.83	1.11	UR
		PUB23	E3 ubiquitin-protein ligase PUB23	1.99	2.49	UR
		PUB9	U-box domain-containing protein 9	1.85	1.20	UR
Osmotic regulation		AGP41	Arabinogalactan protein 41	1.46	1.48	UR
		APF2	Aspartyl protease family protein 2	1.10	2.05	UR
		ASP1	Aspartic proteinase Asp1	1.82	1.65	UR
		At3g56050	Probable inactive receptor-like protein kinase At3g56050	1.28	1.29	UR
		At4g16563	Probable aspartyl protease At4g16563	1.63	1.54	UR
		ERD4	CSC1-like protein ERD4	1.31	1.35	UR
		NTL9	Protein NTM1-like 9 (Calmodulin-binding NAC protein)	1.15	1.37	UR
		PERK1	Proline-rich receptor-like protein kinase PERK1	1.70	2.64	UR
		KRP1	Calcium-binding protein KRP1	3.68	1.78	UR
		RALFL33	Protein RALF-like 33	1.56	1.17	UR
		RXW8	CSC1-like protein RXW8	1.43	2.03	UR
		TPPJ	Probable trehalose-phosphate phosphatase J	2.59	1.16	UR
		TPS7	Probable a-trehalose-phosphate synthase	1.16	1.23	UR
Reactive oxygen scavenging		AAO	L-ascorbate oxidase (ASO)	1.62	2.33	UR
		AO	L-aspartate oxidase	1.04	1.21	UR
		At3g08610	NADH dehydrogenase [ubiquinone]	-1.98	-2.36	DR
		Bp10	L-ascorbate oxidase homolog	3.55	4.50	UR
		CYP76A2	Cytochrome P450 76A2	1.29	2.51	UR
		SDR2a	Short-chain dehydrogenase reductase 2a	2.15	2.08	UR
Regulation of other	Aquaporin	SYP121	Protein Syntaxin of plants 121	2.70	1.89	UR
functional proteins	Response protein	At2g40140	Zinc finger CCCH domain-containing protein 29	1.78	1.01	UR
		At4g27290	G-type lectin S-receptor-like serine/threonine-protein kinase At4g27290	2.32	1.25	UR
		ATL6	E3 ubiquitin-protein ligase ATL6	1.18	1.46	UR
		BAP2	BON1-associated protein 2	2.30	1.54	UR
		DGK1	Diacylglycerol kinase 1	1.01	1.49	UR
		DGK2	Diacylglycerol kinase 2	2.64	2.19	UR
		FLS2	LRR receptor-like serine/threonine-protein kinase FLS2	1.41	2.93	UR
		GATA8	GATA transcription factor 8	2.80	1.17	UR
		NCL	Sodium/calcium exchanger NCL	2.59	2.69	UR
		NHL10	NDR1/HIN1-like protein 10	1.55	1.36	UR
		NHL13	NDR1/HIN1-like protein 13	2.32	1.02	UR
		PBL3	Probable serine/threonine-protein kinase PBL3	1.56	1.51	UR
		PCR2	Protein plant cadmium resistance 2	1.56	1.58	UR
		PCRK1	Serine/threonine-protein kinase PCRK1	1.24	1.01	UR
		RPV1	Disease resistance protein RPV1	2.22	2.17	UR
		WRKY40	Probable WRKY transcription factor 40	1.58	2.49	UR
		WRKY53	Probable WRKY transcription factor 53	1.13	1.53	UR
	Cell wall construction	CDC20-1	Cell division cycle 20.1, cofactor of APC complex	1.92	1.23	UR
		CESA2	Cellulose synthase A catalytic subunit 2	3.21	2.21	UR
		CHIT3	Hevamine-A	1.48	1.13	UR
		CSLC12	Probable xyloglucan glycosyltransferase 12	1.14	1.27	UR
		DCR	BAHD acyltransferase DCR	1.65	1.82	UR
		MUCI21	Xylan glycosyltransferase MUCI21	1.66	1.51	UR
		TBR	Protein trichome birefringence	1.39	1.07	UR
		THE1	Receptor-like protein kinase THESEUS 1	1.67	1.08	UR
		XTH15	Xyloglucan endotransglucosylase/hydrolase 15	2.59	1.73	UR
		XTH22	Xyloglucan endotransglucosylase/hydrolase protein 22	2.85	1.27	UR
		XTH23	Probable xyloglucan endotransglucosylase/hydrolase protein 23	3.07	2.35	UR
		XXT3	Probable xyloglucan 6-xylosyltransferase 3	1.60	1.66	UR
		XXT5	Probable xyloglucan 6-xylosyltransferase 5	1.63	1.26	UR

两品种当归转录组分析及抗旱关键基因挖掘

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康舒淇 ¹ , 朱田田 ¹^,²^,³^,⁴^,^* , 晋玲 ¹^,²^,³^,⁴ , 刘天乐 ¹ , 张菁 ¹ , 张明惠 ¹ , 徐丽 ¹ , 张帅 ¹

中国药学杂志 | 论著 2024,59(12): 1100-1109

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中国药学杂志 | 论著 2024, 59(12): 1100-1109

两品种当归转录组分析及抗旱关键基因挖掘

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康舒淇¹, 朱田田¹^,²^,³^,⁴^,^*, 晋玲¹^,²^,³^,⁴, 刘天乐¹, 张菁¹, 张明惠¹, 徐丽¹, 张帅¹

作者信息

¹ 甘肃中医药大学药学院, 兰州 730000

² 西北中藏药省部共建协同创新中心, 兰州 730000

³ 甘肃省珍稀中药资源评价与保护利用工程研究中心, 兰州 730000

⁴ 陇药产业创新研究院, 兰州 730000

康舒淇,女,硕士研究生研究方向:中药资源保护、评价与可持续利用研究

通讯作者:

*朱田田,女,副教授,硕士生导师研究方向:中药资源评价与分子生药学研究 Tel:(0931)5161169

Transcriptome Analysis of Two Cultivars of Angelica sinensis and Excavation of Key Gene for Drought Resistance

Shuqi KANG¹, Tiantian ZHU¹^,²^,³^,⁴^,^*, Ling JIN¹^,²^,³^,⁴, Tianle LIU¹, Jing ZHANG¹, Minghui ZHANG¹, Li XU¹, Shuai ZHANG¹

Affiliations

¹ College of Pharmacy, Gansu University of Traditional Chinese Medicine, Lanzhou 730000, China

² Northwest Collaborative Innovation Center for Traditional Chinese Medicine Coconstructed by Gansu Province and MOE of PRC, Lanzhou 730000, China

³ Engineering Research Center for Evaluation, Protection, and Utilization of Rare Traditional Chinese Medicine Resources, Lanzhou 730000, China

⁴ Gansu Pharmaceutical Industry Innovation Research Institute, Lanzhou 730000, China

出版时间: 2024-06-22 doi: 10.11669/cpj.2024.12.005

文章导航

摘要

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目的对当归[Angelica sinensis (Oliv.) Diels]主栽品种“岷归1号”和“岷归2号”进行转录组测序差异比较分析,挖掘当归响应干旱胁迫关键基因。方法以两品种当归的新鲜叶片和根组织为研究材料构建cDNA文库,利用二代高通量测序平台Illumina HiSeq^TM 4000进行测序分析,从差异表达基因中筛选响应干旱胁迫的关键基因。结果转录组测序共获得584 423 236条高质量序列,Q20(碱基量≥20%)与Q30(碱基量≥30%)占比分别在97.47%、92.64%以上,鸟嘌呤和胞嘧啶所占比例(GC)含量是42.78%~43.15%。两品种当归叶片和根中共筛选得到1 894个差异表达基因(DEGs),数目分别为674和1 220个,共有差异表达基因为338个。基因本体(GO)富集结果表明,两品种当归在同一组织部位中的DEGs注释分类主要包括细胞过程、代谢过程和催化活性等功能。京都数据库与基因组百科全书(KEGG)分析发现差异表达基因均在植物-病原互作、植物-MAPK信号通路、苯丙烷生物合成和植物激素信号转导等通路中显著富集,精细分类注释结果与GO及KEGG分析结果趋势相符合。基于功能注释结果,挖掘到抗旱相关基因60个,选取HVA22C、KRP1、PUB23、DREB1B、Bp10通过实时荧光定量聚合酶链反应(qRT-PCR)验证其表达量,结果表明,其基因表达水平与转录组测序基因表达趋势一致。结论两品种当归在脱落酸调节、渗透调节、活性氧清除和其他功能蛋白质调节等抗旱途径方面存在一定差异性,筛选出的抗旱基因可为进一步研究当归响应干旱胁迫的分子机制提供数据参考。

关键词

当归 / 品种 / 差异表达基因分析 / 抗旱基因 / 实时荧光定量聚合酶链反应

Abstract

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OBJECTIVE To excavate the key genes of drought stress response of A.sinensis, and carry out the comparative analysis with the transcriptome data of the main cultivars ‘Mingui 1’ and ‘Mingui 2’ of Angelica sinensis (Oliv.) Diels. METHODS With the fresh leaf and root tissues of two cultivars of A.sinensis as materials, a cDNA library was constructed. The Illumina HiSeq^TM 4000, a second-generation high-throughput sequencing platform, was used for sequencing analysis, and key enzyme genes in response to drought stress were screened from differentially expressed genes(DEGs). RESULTS A total of 584 423 236 clean reads were obtained from transcriptome sequencing, in which the percentage of Q20 (base amount ≥20%) and Q30 (base amount ≥30%) were above 97.47% and 92.64%, and the GC content ranged from 42.78% to 43.15%. A total of 1 894 DEGs were screened from the leaves and roots of two cultivars of A.sinensis, the numbers of which were 674 and 1 220, respectively, and they had 338 shared DEGs. The GO enrichment results showed that the annotation classification of DEGs of two cultivars of A.sinensis in the same tissue part mainly included cellular process, metabolic process and catalytic activity. KEGG analysis found that the DEGs were significantly enriched in plant-pathogen interaction, MAPK signaling pathway-plant, phenylpropanoid biosynthesis and plant hormone signal transduction pathways. The detailed classification annotation results were consistent with the trends of GO and KEGG analysis. Based on the functional annotation results, 60 drought resistance genes were excavated. HVA22C, KRP1, PUB23, DREB1B and Bp10 were selected to verify their expression levels by qRT-PCR. and the results showed their genes expression level were consistent with the transcriptome sequencing gene expression trends. CONCLUSION The two cultivars of A.sinensis have some differences in drought resistance pathways such as abscisic acid regulation, osmoregulation, scavenging of reactive oxygen species and regulation of other functional proteins, and the screened drought resistance genes can provide data references for further research on the molecular mechanisms of A.sinensis in response to drought stress.

Key words

A. sinensis / cultivar / DEGs analysis / drought resistance gene / qRT-PCR

引用本文

康舒淇, 朱田田, 晋玲, 刘天乐, 张菁, 张明惠, 徐丽, 张帅. 两品种当归转录组分析及抗旱关键基因挖掘. 中国药学杂志, 2024 , 59 (12) : 1100 -1109 . DOI: 10.11669/cpj.2024.12.005

Shuqi KANG, Tiantian ZHU, Ling JIN, Tianle LIU, Jing ZHANG, Minghui ZHANG, Li XU, Shuai ZHANG. Transcriptome Analysis of Two Cultivars of Angelica sinensis and Excavation of Key Gene for Drought Resistance[J]. Chinese Pharmaceutical Journal, 2024 , 59 (12) : 1100 -1109 . DOI: 10.11669/cpj.2024.12.005

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当归为伞形科植物当归[Angelica sinensis (Oliv.) Diels]的干燥根,是我国传统大宗中药材之一,具有补血活血、调经止痛、润燥通肠的功效^[1]。当归目前以栽培品种为主,产区主要分布在甘肃、云南、四川等省^[2],其中甘肃岷县种植历史悠久、产量大,药材质量优,为著名的道地药材主产区^[3]。“岷归1号”和“岷归2号”是通过系统育种法选育出的优良品种,“岷归1号”茎呈紫色,为目前种植区域最大的品种,“岷归2号”茎呈绿色,具高产稳产、抗逆能力强等特点,是近年来被逐步推广种植的品种^[4-5]。研究发现两品种当归茎叶色差异与类黄酮代谢物有关,而此类化合物在植物发育和防御响应方面也存在重要作用^[6]。在抗性适应能力方面,“岷归1号”对辐照胁迫敏感性较强,但“岷归2号”对其耐受性更强^[7]。

外界环境给予的各种生物和非生物胁迫都会对药用植物造成一定负面影响,其中干旱作为极易发生的胁迫之一,可能会严重制约植物生长发育^[8]。在当归种植过程中,除了抽薹开花及根际土壤病原微生物导致连作障碍等问题会对其产量和品质造成影响^[9-10],近年来全球气候变暖、极端气候等问题的出现均导致干旱风险增加,且栽培环境属西北干旱和半干旱气候区,故干旱也是限制当归生长发育及产量的胁迫因素之一^[11],会使得植物叶片萎蔫、相对含水量降低及活性氧代谢失常等,进而导致氧化损伤至死亡;同时干旱胁迫也可能改变植物内源性激素合成代谢、信号转导通路及各种生物代谢调控等,进而影响药材质量^[12]。当前对当归的研究主要集中于种质资源评价、药效成分和药理作用研究等方面^[13],针对当归的干旱胁迫还未见系统性研究。

课题组前期对干旱胁迫后的两品种当归植株形态做差异对比,发现在同一程度干旱胁迫下,“岷归1号”叶片卷曲程度及植株下垂状况较“岷归2号”更为明显,推测“岷归2号”抗旱适应能力可能略强于“岷归1号”(图1 A)。本研究通过对两品种当归“岷归1号”和“岷归2号”进行转录组测序比较分析,从中挖掘当归抗旱关键基因,可为研究当归响应干旱胁迫的分子机制提供候选基因,旨在为当归药材生态种植评价、品质提升及耐旱性品种培育提供研究基础。

1 材料

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1.1 植物材料

当归全长转录组参考序列数据已上传于国家生物技术信息中心(NCBI)(Access:PRJNA782300)^[6];选取同一时间种植且生境条件相同的成药生长期“岷归1号”和“岷归2号”新鲜叶片、根组织为研究材料,取样部位为顶端叶片及根部膨大处(图1B),样品均于2022年9月18日采自甘肃省定西市临洮县(海拔2 708 m;35°37'52.22''N,104°7'10.13''E)。经甘肃中医药大学中药资源教研室晋玲教授鉴定为伞形科植物当归[A. sinensis (Oliv.) Diels]。样本组织采集后用水清洗干净,投入液氮中带回实验室置于-80 ℃超低温冰箱保存,每个组织样品设置3个生物学重复,用于转录组分析及差异表达基因验证,样品编号信息见表1。

1.2 仪器与试剂

1.2.1 仪器

FTC-3000P 荧光定量PCR仪(加拿大 FunglynBiotech 公司);DYY-7C 微型转移电泳仪(北京六一仪器厂);c300 多功能分子成像系统(美国 Azure Biosystems 公司);MYSPIN6型台式高速冷冻离心机(美国Thermo FisherScientific公司);Tone 96g 梯度聚合酶链式反应(PCR) 扩增仪(德国analytik-jena公司);EXF40086V 超低温冰箱(北京五洲东方科技发展有限公司)。

1.2.2 试剂

Kit R6827 植物RNA提取试剂盒(美国Omega公司);FastKing RT Kit-KR116试剂盒(北京天根生化科技有限公司);合成引物(上海生工生物工程股份有限公司);Q341型逆转录聚合酶链式反应(RT-PCR)试剂盒(南京诺唯赞生物科技股份有限公司)。

2 方法

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2.1 总RNA提取、cDNA文库构建及RNA-seq测序

采用Trizol法提取两品种当归叶片、根组织总RNA, Agilent 2100 Bioanalyzer检测总RNA样品的质量,并使用琼脂糖凝胶电泳检验是否存在污染,检验合格的RNA样品用于cDNA文库的构建。将mRNA打断为片状并以之为模板反转录为双链cDNA,QiaQuick-PCR试剂盒纯化片段,经修复末端,加A尾连接测序接头,筛选片段进行PCR扩增以完成当归叶片及根组织样品文库构建。最后,利用Gene Denovo生物技术公司的Illumina HiSeq^TM 4000进行测序分析。

2.2 测序数据质量过滤、组装及基因表达分析

利用fastp^[14](Version 0.18.0)将原始序列数据(raw reads)过滤得到质量值Q20(碱基量≥20%)与Q30(碱基量≥30%)合格、鸟嘌呤和胞嘧啶所占比例(GC)含量及分布符合预期的高质量序列(clean reads);clean reads使用Trinity软件^[15]进行拼接组装获得unigenes;以课题组前期实验得到的全长转录本为参考,运用RSEM(Version 1.2.19)软件^[16]对各样品进行基因表达定量,相对表达水平以RPKM值(reads per kilobase per million mapped reads)表示,计算出的基因表达量可直接比较两品种当归间基因表达差异,用于后续分析。

2.3 差异表达基因分析

使用DESeq2(version 1.20.0)^[17]软件对两品种当归同一组织部位进行转录水平差异比较,本研究设置2个差异组别,叶片组织为M1L vs M2L,根组织为M1R vs M2R,以|log₂(Fold-Change)|>1和错误发现率(false discovery rate,FDR)<0.05为差异表达基因(differentially expressed genes,DEGs)筛选条件。基于筛选结果,通过基因本体(Gene Ontology,GO)数据库与京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库对DEGs进行GO功能分类统计和KEGG通路富集分析^[18-19]。为了进一步挖掘注释DEGs种类功能,将DEGs在Swiss-Prot(Swissprot Protein Sequence Database)数据库中比对,利用Uniprot(Universal Protein)数据库对已鉴定和去重后DGEs的生物学功能和分子功能进行精细分类统计。

2.4 实时荧光定量聚合酶链式反应(qRT-PCR)表达量验证

为明确转录组数据的可靠性,基于差异分析结果,选取5个关键DGEs (HVA22C、KRP1、PUB23、DREB1B、Bp10)进行qRT-PCR验证。运用NCBI Primer-BLAST网站设计引物,委托生工生物工程(上海)股份有限公司合成,引物详情见表2。研究选用ACT(actin)作为内参基因^[20],根据诺唯赞RT-PCR试剂盒(Q341)说明书配制反应体系及设置反应程序以完成qRT-PCR检测,体系含2×ChamQ SYBR qPCR Master Mix 10 μL,正、反引物各 0.4 μL,双蒸H₂O 5.2 μL,cDNA模板4 μL,总体积为20 μL;反应程序为95 ℃ 90 s,95 ℃ 5 s,60 ℃ 15 s,72 ℃ 20 s,40个循环。采用2^-△△Ct法^[21]计算基因相对表达量。

3 结果与分析

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3.1 测序数据质量分析

对两品种当归叶片、根组织进行测序分析,经测序共获得584 423 236条clean reads,均占raw reads的99.26%以上。所有测序数据Q20与Q30占比分别在97.47%、92.64%以上;GC含量范围是42.78%~43.15%;将各样品clean reads进行序列比对,两品种当归叶片和根中分别有136 283 756、130 651 633、113 167 110和113 740 062 条mapped reads可定位到基因上,mapped ratio均在88.68%、87.08%、80.32%及76.07%以上,说明过滤后碱基组成及分布较平衡,数据质控良好,可用于后续准确分析(表3)。

3.2 差异表达基因分析

火山图中,log₂FC>1为表达上调,log₂FC<-1则为表达下调。由图2A和B可见,M1L vs M2L中上调表达基因(up-regulated,UR)553个,下调表达基因(down-regulated,DR)121个;M1R vs M2R中UR 1 069个,DR 151个,共有DEGs中UR 324个,DR 14个,说明相同生态环境下的M1和M2差异表达较大,同一组织部位中UR的数量略多于DR。基于筛选条件,共检测获得1 894个DEGs,分布于叶片中有674个DEGs,根中有1 220个,其中338个为共有DEGs(图2C)。

3.2.1 GO富集分析

基于GO数据库注释基因功能,比较不同差异组别间的差异倍数,将差异基因集(M1L vs M2L、M1R vs M2R)比对注释于参与生物过程(biological process)、细胞组分(cellular component)、分子功能(molecular function)3个大类别。通过GO功能富集分析,可进一步明确两品种当归DEGs主要集中哪些功能类别。GO富集分析结果显示(图3),M1L vs M2L与M1R vs M2R全部DEGs分为42和46种功能亚类别,其中参与生物过程在两差异组别注释数目为20和22种,主要分布于细胞过程(cellular process)、代谢过程(metabolic process)和单一有机体过程(single-organism process),M1L vs M2L中UR与DR数目分别为325、281、294及32、41、34个,M1R vs M2R中UR与DR数目分别为634、582、554及63、63、58个。两差异组别注释于细胞组分数目为13和15种,排名前三的为细胞(cell)、细胞部分(cell part)及细胞器(organelle),M1L vs M2L中UR与DR数目分别为278、276、219及33、33、25个,M1R vs M2R中UR与DR数目分别为478、473、382及59、58、52个。两差异组别比对到分子功能的富集数目均为9种,主要集中在催化活性(catalytic activity)、结合蛋白(binding)及转运活性(transporter activity)中,M1L vs M2L中UR与DR数目分别为179、178、30及36、25、5个,M1R vs M2R中UR与DR数目分别为432、410、92及36、52、2个。

3.2.2 KEGG代谢通路富集分析

根据KEGG分类与注释结果(图4),M1L vs M2L与M1R vs M2R富集通路共分为5类,涉及通路数量由多到少分别是代谢(metabolism)、遗传信息处理(genetic information processing)、环境信息处理(environmental information processing)、细胞过程(cellular processes)和有机系统(organismal systems)。M1L vs M2L与M1R vs M2R参与代谢通路(pathway)分别为56和63条,根据Q-value(FDR校正后P值)筛选出前20个通路,其中主要包括植物-病原互作(plant-pathogen interaction)、植物-丝裂原活化蛋白激酶(MAPK)信号通路(MAPK signaling pathway-plant)、苯丙烷生物合成(phenylpropanoid biosynthesis)、植物激素信号转导(plant hormone signal transduction)、氧化磷酸化(oxidative phosphorylation)、淀粉与蔗糖代谢(starch and sucrose metabolism)以及次生代谢物的生物合成(biosynthesis of secondary metabolites)等,其余通路中DEGs参与较少。

3.2.3 差异表达基因精细分类功能注释

基于Swiss-Prot数据库比对结果,通过Uniprot蛋白质数据库对筛选获得的M1L vs M2L与M1R vs M2R差异基因转录数据进行精细分类功能注释。两差异组别各包含411和715个重复基因,剩余已鉴定基因数目分别为263和505个。在已鉴定DEGs中,M1L vs M2L中有26个注释无生物学特性,M1R vs M2R则为25个,根据生物学功能和分子功能可将237和480个DEGs注释为12类:光合作用(photosynthesis,9,10)、呼吸作用(respiration,9,10)、初级代谢(primary metabolism,35,82)、次生代谢(secondary metabolism,11,29)、激素生物合成(hormone biosynthesis,1,10)、生物信号(bio-signaling,25,67)、多核苷酸生物合成(polynucleotide biosynthesis,6,18)、细胞形态发生(cell morphogenesis,36,46)、转录因子(transcription factors,33,61)、翻译(translation,6,22)功能、转运(transport,21,47)功能以及逆境响应(stress response,45,77),见图5。M1L vs M2L精细功能分类占比居前五位的是代谢、逆境响应、细胞形态发生、转录因子和生物信号,比例依次为19.41%、18.99%、15.19%、13.92%和10.55%。M1R vs M2R主要分布于代谢、逆境响应、生物信号、转录因子和转运功能,比例依次为23.12%、16.94%、13.96%、12.71%和9.79%,注释结果与GO及KEGG功能富集结果趋势相符合。

3.2.4 抗旱相关差异表达基因筛选

根据功能注释及富集代谢途径结果对两差异组别中相同DEGs进一步精细筛选,筛选得到有关脱落酸(ABA)调节、渗透调节、活性氧清除和其他功能蛋白质调节的抗旱相关基因共60个。其中属于DREB转录因子亚家族的关键基因主要包括DREB1B、DREB1D,在当归叶片和根中均呈上调表达模式,表明该类基因在M2中上调,且DREB1B在根中表达量略高于叶片,DREB1D则在叶片中高表达。脱水诱导早期反应ERD基因(ERD7、ERD15)参与胁迫相关激素诱导,在叶片中的表达量高于根中。涉及ABA信号转导途径的HVA22基因(HVA22A、HVA22C)中,HVA22A在叶片和根中表达量相近,log₂FC值分别为1.82和1.84,HVA22C则在根中显著高表达,log₂FC值分别为1.94和2.87。植物-MAPK信号通路中的丝裂原激活蛋白激酶3(MPK3)基因具有调节气孔细胞呼吸的功能,在叶片中显著高表达。U-box基因家族E3泛素蛋白连接酶(PUB9、PUB23)可协同调控干旱信号通路,PUB9在叶片中表达量高于根中,PUB23则在根中表达量高于叶中。参与渗透调节的关键基因主要包括阿拉伯半乳蛋白(AtAGP41)、天冬氨酸蛋白酶(APF2、ASP1、At3g56050、At4g16563)、钙信号蛋白(ERD4、RXW8、KRP1)、脯氨酸受体样蛋白激酶(PERK1)、海藻糖-6-磷酸磷酸酶(TPPJ)及海藻糖-6-磷酸合成酶(TPS7),其基因表达量在叶片和根中均呈不同程度上调。氧化还原关键酶包括2个抗坏血酸氧化酶(AAO、Bp10)、2个脱氢酶还原酶(At3g08610、SDR2a)、1个氧化还原酶(CYP76A2)、1个天冬氨酸氧化酶(AO),除At3g08610基因表达下调之外,其余基因均表达上调。从差异表达量角度分析,筛选出的抗旱相关基因在叶片和根组织差异组中均呈现M2大于M1的情况,且上调DEGs多于下调DEGs,只有少数筛选基因下调,结果表明,M2的抗旱性可能略强于M1(表4)。

3.3 差异表达基因的qRT-PCR验证

根据当归转录组测序结果对部分关键基因进行定量分析,随机选取抗旱相关基因HVA22C、KRP1、PUB23、DREB1B、Bp10进行qRT-PCR验证(n=3)。结果见图6,5个关键基因在两品种当归叶片和根中表达变化情况与转录组测序基因表达趋势一致,说明本研究测序数据可靠性较高,参考性较强。

4 讨论

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干旱是影响植物生长发育的主要限制因素之一,植物响应干旱胁迫是一个复杂信号调控过程,其转录网络受多个基因调节,涉及多种代谢产物及合成途径。利用转录组测序技术可使基因表达量化,挖掘关键功能基因,是培育品种优良性状和研究基因功能的重要手段^[22]。本研究通过对两品种当归进行转录组测序,结合GO、KEGG和精细功能注释对筛选获得的DEGs分类,发现差异功能主要集中于生长代谢、抗逆响应、苯丙烷类、黄酮类等化合物生物合成及各类生物反应等过程,推测两品种当归可能在生长发育、抗胁迫能力和药效成分合成等方面均存在一定差异性。

当前研究干旱胁迫的调节途径主要包括ABA调节、渗透调节、活性氧清除和其他功能蛋白质调节等,涉及脱落酸、过氧化氢酶等多种物质的合成、代谢及信号转导通路^[23]。部分转录因子(transcription factor,TF)可通过结合特异元件来调节功能基因表达以介导非生物胁迫信号转导。DREB转录因子属于AP2/ERF转录因子家族的一个亚家族,该亚家族基因可增强抗逆能力^[24]。ERD基因最早发现于拟南芥脱水诱导,也受其他低温、高盐、ABA等胁迫影响,ERD15是ABA反应和依赖水杨酸(SA)防御途径的重要调节器^[25]。HVA22家族基因在ABA和应激诱导下,编码一类具有保守TB2/DP1/HVA22结构域的应激反应蛋白,可结合干旱响应(MYB)防御和胁迫响应元件(TC-rich repeats)以及激素响应元件(ABRE、ERE、SARE等)^[26]。PUB9、PUB23属于植物U-box E3泛素蛋白连接酶,对干旱胁迫表现出超敏性,泛素化可能会改善植株抗旱能力^[27]。在当归转录数据中,以上关键基因在叶片和根中均为上调表达模式,表明其可能正向调控当归抗旱过程,响应ABA调节引起叶片气孔关闭,直接性降低蒸腾作用导致的水分流失,有效影响当归抵御干旱并提高其抗旱性。

渗透调节也是药用植物抵抗干旱的重要生理机制之一,本研究涉及的渗透调节物质包括脯氨酸(Pro)、可溶性碳水化合物、钙信号蛋白等。耐旱植物可通过积累蔗糖、海藻糖等来提高抗旱性,海藻糖的主合成途径为海藻糖-6-磷酸合成酶-海藻糖-6-磷酸磷酸酶(trehalose-6-phosphate synthase-trehalose-6-phosphate phosphatase,TPS-TPP)途径,TPS与TPP受环境诱导表达,促使代谢物生成,目前已在多个物种间被鉴定^[28]。ERD4、RXW8、KRP1等钙信号蛋白基因在当归叶片和根中呈现上调表达模式,表明其跨膜转导作用可调节相对含水量以维持细胞渗透压,增加渗透势,以此反映抗旱性^[29]。药用植物在干旱胁迫下可产生有害活性氧,抗氧化系统可发挥抵御或保护水分能力,而本研究中抗氧化酶基因AAO、Bp10、At3g08610、SDR2a、CYP76A2、AO的上调表达也在一定程度说明其具有清除活性氧功能,以此在干旱胁迫中达到动态平衡抵御干旱的作用^[30]。

此外,还有其他功能蛋白质也可能参与干旱胁迫,在当归共有DEGs中,涉及分类主要包括水通道蛋白、响应蛋白及细胞壁建构等,基因数目分别为1、17和13个。水通道蛋白可通过增加细胞膜通透性来进行高效物质转运,从而提高对各种胁迫的耐受性,SYP121在当归叶片中表达量显著高于根中,说明该基因可能在叶片中响应胁迫程度大于根中;有学者对植株干旱胁迫发现,其叶片抗病基因与抗旱基因表达模式一致^[31],本研究中各类响应蛋白,如发育蛋白基因(At4g27290、WRKY53)、抗病蛋白基因(ATL6、BAP2、FLS2、NHL10、NHL13、PBL3、PCRK1、RPV1、WRKY40)、盐胁迫蛋白基因(At2g40140、NCL)、冷胁迫蛋白基因(DGK1、DGK2)及解毒蛋白基因(PCR2)等均与直接参与抗旱的基因表达一致,间接反映以上基因可能与抗旱性存在交叉协同调控作用;植物细胞壁建构合成与木质素、角质、蜡质合成相关,CESA2、CHIT3、CSLC12、DCR等基因通过调控变化促使植物表层形成屏障,与耐旱性形成有一定联系,且在药用植物生长发育和抗性方面发挥显著作用。

本研究对相同生境条件下的两品种当归进行转录比较,经qRT-PCR初步筛选验证抗旱关键基因HVA22C、KRP1、PUB23、DREB1B、Bp10,为研究当归响应干旱胁迫的分子机制提供候选基因。同时,此结果也为后续进一步探究当归基因功能验证提供基因资源。

基金

收起

甘肃省高校青年博士基金项目资助(2023QB-094)
甘肃省科技重大专项资助(23ZDFA013-1)
甘肃省教育厅“双一流”科研重点项目资助(GSSYLXM-05)
甘肃省科技计划项目资助(20JR5RA182)
甘肃中医药大学科学研究与创新基金项目资助(2021KCZD-4)
西北中藏药省部共建协同创新中心开放基金项目资助(Xbzzy202207)

参考文献

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2024年第59卷第12期

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文章信息

doi: 10.11669/cpj.2024.12.005

接收时间：2023-10-09
首发时间：2025-11-25
出版时间：2024-06-22

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文章信息

作者

出版历史

收稿日期：2023-10-09

基金

甘肃省高校青年博士基金项目资助(2023QB-094)

甘肃省科技重大专项资助(23ZDFA013-1)

甘肃省教育厅“双一流”科研重点项目资助(GSSYLXM-05)

甘肃省科技计划项目资助(20JR5RA182)

甘肃中医药大学科学研究与创新基金项目资助(2021KCZD-4)

西北中藏药省部共建协同创新中心开放基金项目资助(Xbzzy202207)

作者信息

¹ 甘肃中医药大学药学院, 兰州 730000

² 西北中藏药省部共建协同创新中心, 兰州 730000

³ 甘肃省珍稀中药资源评价与保护利用工程研究中心, 兰州 730000

⁴ 陇药产业创新研究院, 兰州 730000

通讯作者:

*朱田田,女,副教授,硕士生导师研究方向:中药资源评价与分子生药学研究 Tel:(0931)5161169

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Cultivars name	Tissue site	Sample number(n=3)
Mingui 1(M1)	Leaf	M1L-1、M1L-2、M1L-3
	Root	M1R-1、M1R-2、M1R-3
Mingui 2(M2)	Leaf	M2L-1、M2L-2、M2L-3
	Root	M2R-1、M2R-2、M2R-3

Cultivars name

Tissue site

Sample number(n=3)

Mingui 1(M1)

Leaf

M1L-1、M1L-2、M1L-3

Root

M1R-1、M1R-2、M1R-3

Mingui 2(M2)

Leaf

M2L-1、M2L-2、M2L-3

Root

M2R-1、M2R-2、M2R-3

Gene name	Primer sequence (5-3)	Amplification length/bp
HVA22C	F:TTCAACGGAGCTAGTATTGCCTACG	175
	R:TCTCCAAAGCCTCAGTTCCATTCTC
KRP1	F:GGTGATGTGAATGGAGATGGAGTTC	106
	R:CTTCTTCAAGCCAAACCTCAGATTC
PUB23	F:ACGGCATCCAAAGAATCCCAACTC	175
	R:TAACCCGCTCCTGCTGCTTCC
DREB1B	F:GTGTTGCCAGCTTCCAGTAATCC	93
	R:TCTACGCCTCACTCCTCTGTAAAC
Bp10	F:TCCAAGACCCAACCCACAAGG	147
	R:AGAGGTGTATCCGATGGTATGAAGG
ACT	F:TGGTATTGTGCTGGATTCTGGT	109
	R:TGAGATCACCACCAGCAAGG

Gene
name

Primer sequence
(5-3)

Amplification
length/bp

HVA22C

F:TTCAACGGAGCTAGTATTGCCTACG

175

R:TCTCCAAAGCCTCAGTTCCATTCTC

KRP1

F:GGTGATGTGAATGGAGATGGAGTTC

106

R:CTTCTTCAAGCCAAACCTCAGATTC

PUB23

F:ACGGCATCCAAAGAATCCCAACTC

175

R:TAACCCGCTCCTGCTGCTTCC

DREB1B

F:GTGTTGCCAGCTTCCAGTAATCC

R:TCTACGCCTCACTCCTCTGTAAAC

Bp10

F:TCCAAGACCCAACCCACAAGG

147

R:AGAGGTGTATCCGATGGTATGAAGG

ACT

F:TGGTATTGTGCTGGATTCTGGT

109

R:TGAGATCACCACCAGCAAGG

No.	Raw reads	Clean reads/%	Clean reads/bp	Q20/%	Q30/%	GC/%	Mapped reads	Mapped ratio/%
M1L-1	45 403 718	45 123 184(99.38)	6 673 603 863	97.97	93.75	42.83	40 424 439	89.59
M1L-2	56 479 970	56 136 434(99.39)	8 310 128 766	97.79	93.39	42.95	50 542 876	90.04
M1L-3	51 450 794	51 103 374(99.32)	7 610 806 624	97.64	93.00	42.91	45 316 441	88.68
M2L-1	49 972 658	49 665 860(99.39)	7 374 089 668	97.87	93.53	43.14	44 735 242	90.07
M2L-2	47 873 956	47 583 270(99.39)	7 024 011 837	97.91	93.66	43.10	41 434 239	87.08
M2L-3	49 948 026	49 632 084(99.37)	7 375 539 235	97.47	92.64	42.93	44 482 152	89.62
M1R-1	46 193 770	45 851 916(99.26)	6 790 909 357	97.60	92.96	43.15	37 046 874	80.80
M1R-2	48 730 572	48 436 458(99.40)	7 068 058 117	97.95	93.82	43.04	38 902 366	80.32
M1R-3	46 580 816	46 266 062(99.32)	6 862 726 173	97.69	93.13	42.91	37 217 870	80.44
M2R-1	43 229 068	42 923 476(99.29)	6 341 975 820	97.58	92.88	43.01	34 686 284	80.81
M2R-2	55 754 104	55 400 420(99.37)	8 232 259 847	97.58	92.90	42.78	42 144 671	76.07
M2R-3	46 570 526	46 300 698(99.42%)	6 884 153 864	97.69	93.10	42.95	36 909 107	79.72

No.

Raw reads

Clean reads/%

Clean reads/bp

Q20/%

Q30/%

GC/%

Mapped reads

Mapped ratio/%

M1L-1

45 403 718

45 123 184(99.38)

6 673 603 863

97.97

93.75

42.83

40 424 439

89.59

M1L-2

56 479 970

56 136 434(99.39)

8 310 128 766

97.79

93.39

42.95

50 542 876

90.04

M1L-3

51 450 794

51 103 374(99.32)

7 610 806 624

97.64

93.00

42.91

45 316 441

88.68

M2L-1

49 972 658

49 665 860(99.39)

7 374 089 668

97.87

93.53

43.14

44 735 242

90.07

M2L-2

47 873 956

47 583 270(99.39)

7 024 011 837

97.91

93.66

43.10

41 434 239

87.08

M2L-3

49 948 026

49 632 084(99.37)

7 375 539 235

97.47

92.64

42.93

44 482 152

89.62

M1R-1

46 193 770

45 851 916(99.26)

6 790 909 357

97.60

92.96

43.15

37 046 874

80.80

M1R-2

48 730 572

48 436 458(99.40)

7 068 058 117

97.95

93.82

43.04

38 902 366

80.32

M1R-3

46 580 816

46 266 062(99.32)

6 862 726 173

97.69

93.13

42.91

37 217 870

80.44

M2R-1

43 229 068

42 923 476(99.29)

6 341 975 820

97.58

92.88

43.01

34 686 284

80.81

M2R-2

55 754 104

55 400 420(99.37)

8 232 259 847

97.58

92.90

42.78

42 144 671

76.07

M2R-3

46 570 526

46 300 698(99.42%)

6 884 153 864

97.69

93.10

42.95

36 909 107

79.72

Sort	Subclassification	Gene abbreviation	Comment name	log₂FC	Expression pattern
Abscisic acid regulation		DREB1B	Dehydration-responsive element-binding protein 1B	2.96	3.03	UR
		DREB1D	Dehydration-responsive element-binding protein 1D	2.82	2.30	UR
		ERD7	Protein early-responsive to dehydration 7	1.58	1.18	UR
		ERD15	Protein early-responsive to dehydration 15	1.54	1.41	UR
		HVA22A	HVA22-like protein a	1.82	1.84	UR
		HVA22C	HVA22-like protein c	1.94	2.87	UR
		MPK3	Mitogen-activated protein kinase 3	2.16	2.02	UR
		FER	Receptor-like protein kinase FERONIA	1.83	1.11	UR
		PUB23	E3 ubiquitin-protein ligase PUB23	1.99	2.49	UR
		PUB9	U-box domain-containing protein 9	1.85	1.20	UR
Osmotic regulation		AGP41	Arabinogalactan protein 41	1.46	1.48	UR
		APF2	Aspartyl protease family protein 2	1.10	2.05	UR
		ASP1	Aspartic proteinase Asp1	1.82	1.65	UR
		At3g56050	Probable inactive receptor-like protein kinase At3g56050	1.28	1.29	UR
		At4g16563	Probable aspartyl protease At4g16563	1.63	1.54	UR
		ERD4	CSC1-like protein ERD4	1.31	1.35	UR
		NTL9	Protein NTM1-like 9 (Calmodulin-binding NAC protein)	1.15	1.37	UR
		PERK1	Proline-rich receptor-like protein kinase PERK1	1.70	2.64	UR
		KRP1	Calcium-binding protein KRP1	3.68	1.78	UR
		RALFL33	Protein RALF-like 33	1.56	1.17	UR
		RXW8	CSC1-like protein RXW8	1.43	2.03	UR
		TPPJ	Probable trehalose-phosphate phosphatase J	2.59	1.16	UR
		TPS7	Probable a-trehalose-phosphate synthase	1.16	1.23	UR
Reactive oxygen scavenging		AAO	L-ascorbate oxidase (ASO)	1.62	2.33	UR
		AO	L-aspartate oxidase	1.04	1.21	UR
		At3g08610	NADH dehydrogenase [ubiquinone]	-1.98	-2.36	DR
		Bp10	L-ascorbate oxidase homolog	3.55	4.50	UR
		CYP76A2	Cytochrome P450 76A2	1.29	2.51	UR
		SDR2a	Short-chain dehydrogenase reductase 2a	2.15	2.08	UR
Regulation of other	Aquaporin	SYP121	Protein Syntaxin of plants 121	2.70	1.89	UR
functional proteins	Response protein	At2g40140	Zinc finger CCCH domain-containing protein 29	1.78	1.01	UR
		At4g27290	G-type lectin S-receptor-like serine/threonine-protein kinase At4g27290	2.32	1.25	UR
		ATL6	E3 ubiquitin-protein ligase ATL6	1.18	1.46	UR
		BAP2	BON1-associated protein 2	2.30	1.54	UR
		DGK1	Diacylglycerol kinase 1	1.01	1.49	UR
		DGK2	Diacylglycerol kinase 2	2.64	2.19	UR
		FLS2	LRR receptor-like serine/threonine-protein kinase FLS2	1.41	2.93	UR
		GATA8	GATA transcription factor 8	2.80	1.17	UR
		NCL	Sodium/calcium exchanger NCL	2.59	2.69	UR
		NHL10	NDR1/HIN1-like protein 10	1.55	1.36	UR
		NHL13	NDR1/HIN1-like protein 13	2.32	1.02	UR
		PBL3	Probable serine/threonine-protein kinase PBL3	1.56	1.51	UR
		PCR2	Protein plant cadmium resistance 2	1.56	1.58	UR
		PCRK1	Serine/threonine-protein kinase PCRK1	1.24	1.01	UR
		RPV1	Disease resistance protein RPV1	2.22	2.17	UR
		WRKY40	Probable WRKY transcription factor 40	1.58	2.49	UR
		WRKY53	Probable WRKY transcription factor 53	1.13	1.53	UR
	Cell wall construction	CDC20-1	Cell division cycle 20.1, cofactor of APC complex	1.92	1.23	UR
		CESA2	Cellulose synthase A catalytic subunit 2	3.21	2.21	UR
		CHIT3	Hevamine-A	1.48	1.13	UR
		CSLC12	Probable xyloglucan glycosyltransferase 12	1.14	1.27	UR
		DCR	BAHD acyltransferase DCR	1.65	1.82	UR
		MUCI21	Xylan glycosyltransferase MUCI21	1.66	1.51	UR
		TBR	Protein trichome birefringence	1.39	1.07	UR
		THE1	Receptor-like protein kinase THESEUS 1	1.67	1.08	UR
		XTH15	Xyloglucan endotransglucosylase/hydrolase 15	2.59	1.73	UR
		XTH22	Xyloglucan endotransglucosylase/hydrolase protein 22	2.85	1.27	UR
		XTH23	Probable xyloglucan endotransglucosylase/hydrolase protein 23	3.07	2.35	UR
		XXT3	Probable xyloglucan 6-xylosyltransferase 3	1.60	1.66	UR
		XXT5	Probable xyloglucan 6-xylosyltransferase 5	1.63	1.26	UR

Sort

Subclassification

Gene
abbreviation

Comment name

log₂FC

Expression
pattern

Leaf

Root

Abscisic acid regulation

DREB1B

Dehydration-responsive element-binding protein 1B

2.96

3.03

DREB1D

Dehydration-responsive element-binding protein 1D

2.82

2.30

ERD7

Protein early-responsive to dehydration 7

1.58

1.18

ERD15

Protein early-responsive to dehydration 15

1.54

1.41

HVA22A

HVA22-like protein a

1.82

1.84

HVA22C

HVA22-like protein c

1.94

2.87

MPK3

Mitogen-activated protein kinase 3

2.16

2.02

FER

Receptor-like protein kinase FERONIA

1.83

1.11

PUB23

E3 ubiquitin-protein ligase PUB23

1.99

2.49

PUB9

U-box domain-containing protein 9

1.85

1.20

Osmotic regulation

AGP41

Arabinogalactan protein 41

1.46

1.48

APF2

Aspartyl protease family protein 2

1.10

2.05

ASP1

Aspartic proteinase Asp1

1.82

1.65

At3g56050

Probable inactive receptor-like protein kinase At3g56050

1.28

1.29

At4g16563

Probable aspartyl protease At4g16563

1.63

1.54

ERD4

CSC1-like protein ERD4

1.31

1.35

NTL9

Protein NTM1-like 9 (Calmodulin-binding NAC protein)

1.15

1.37

PERK1

Proline-rich receptor-like protein kinase PERK1

1.70

2.64

KRP1

Calcium-binding protein KRP1

3.68

1.78

RALFL33

Protein RALF-like 33

1.56

1.17

RXW8

CSC1-like protein RXW8

1.43

2.03

TPPJ

Probable trehalose-phosphate phosphatase J

2.59

1.16

TPS7

Probable a-trehalose-phosphate synthase

1.16

1.23

Reactive oxygen scavenging

AAO

L-ascorbate oxidase (ASO)

1.62

2.33

L-aspartate oxidase

1.04

1.21

At3g08610

NADH dehydrogenase [ubiquinone]

-1.98

-2.36

Bp10

L-ascorbate oxidase homolog

3.55

4.50

CYP76A2

Cytochrome P450 76A2

1.29

2.51

SDR2a

Short-chain dehydrogenase reductase 2a

2.15

2.08

Regulation of other

Aquaporin

SYP121

Protein Syntaxin of plants 121

2.70

1.89

functional proteins

Response protein

At2g40140

Zinc finger CCCH domain-containing protein 29

1.78

1.01

At4g27290

G-type lectin S-receptor-like serine/threonine-protein kinase At4g27290

2.32

1.25

ATL6

E3 ubiquitin-protein ligase ATL6

1.18

1.46

BAP2

BON1-associated protein 2

2.30

1.54

DGK1

Diacylglycerol kinase 1

1.01

1.49

DGK2

Diacylglycerol kinase 2

2.64

2.19

FLS2

LRR receptor-like serine/threonine-protein kinase FLS2

1.41

2.93

GATA8

GATA transcription factor 8

2.80

1.17

NCL

Sodium/calcium exchanger NCL

2.59

2.69

NHL10

NDR1/HIN1-like protein 10

1.55

1.36

NHL13

NDR1/HIN1-like protein 13

2.32

1.02

PBL3

Probable serine/threonine-protein kinase PBL3

1.56

1.51

PCR2

Protein plant cadmium resistance 2

1.56

1.58

PCRK1

Serine/threonine-protein kinase PCRK1

1.24

1.01

RPV1

Disease resistance protein RPV1

2.22

2.17

WRKY40

Probable WRKY transcription factor 40

1.58

2.49

WRKY53

Probable WRKY transcription factor 53

1.13

1.53

Cell wall construction

CDC20-1

Cell division cycle 20.1, cofactor of APC complex

1.92

1.23

CESA2

Cellulose synthase A catalytic subunit 2

3.21

2.21

CHIT3

Hevamine-A

1.48

1.13

CSLC12

Probable xyloglucan glycosyltransferase 12

1.14

1.27

DCR

BAHD acyltransferase DCR

1.65

1.82

MUCI21

Xylan glycosyltransferase MUCI21

1.66

1.51

TBR

Protein trichome birefringence

1.39

1.07

THE1

Receptor-like protein kinase THESEUS 1

1.67

1.08

XTH15

Xyloglucan endotransglucosylase/hydrolase 15

2.59

1.73

XTH22

Xyloglucan endotransglucosylase/hydrolase protein 22

2.85

1.27

XTH23

Probable xyloglucan endotransglucosylase/hydrolase protein 23

3.07

2.35

XXT3

Probable xyloglucan 6-xylosyltransferase 3

1.60

1.66

XXT5

Probable xyloglucan 6-xylosyltransferase 5

1.63

1.26