PEG模拟干旱条件下黄瓜种子发芽前后转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0819

摘要/Abstract

摘要：

干旱是影响黄瓜生长发育并导致产量降低最为普遍的非生物胁迫因素之一。为解析黄瓜干旱胁迫响应分子作用机制，以抗旱型旱黄瓜‘KS33’与敏感型旱黄瓜‘KS30’为试材，利用PEG模拟干旱对种子进行处理，选取发芽前期和发芽后期进行转录组测序。结果表明，发芽前期是抗旱基因挖掘的重要时期，其中，抗旱型材料发芽前期PEG模拟干旱诱导较对照（DT1_TvsDT1_CK）鉴定到727个上调和345个下调差异基因，敏感型材料发芽前期PEG模拟干旱诱导较对照（DS1_TvsDS1_CK）鉴定到1 226个上调和111个下调差异基因；KEGG富集联合分析鉴定到富集于亚油酸代谢（csv00591）、戊糖、葡萄糖醛酸转换（csv00040）、苯丙素的生物合成（csv00940）和植物激素信号转导（csv04075）通路上的20个抗旱关键候选基因。关联SNP变异位点信息及RT-qPCR分析验证，最终筛选出抗旱基因CsaV3_4G023820、CsaV3_2G006420及2个相关SNP分子标记。

关键词: 黄瓜, 干旱, 转录组, 分子标记

Abstract:

Drought is one of the most common abiotic stress factors that affect the growth and development of cucumber and reduce the yield. To analyze the molecular mechanism of cucumber drought stress response, the seeds of drought resistant cucumber ‘KS33’and drought sensitive cucumber ‘KS30’ were treated with PEG drought simulation, and transcriptomic sequencing was performed at early and late germination stages. The results showed that early germination was an important database for drought resistance gene mining, among which 727 up-regulated and 345 down-regulated differential genes were identified in PEG drought simulation induction of drought-resistant materials in early germination compared with the control（DT1_TvsDT1_CK）. Compared with the control（DS1_TvsDS1_CK）, 1 226 up-regulated and 111 down-regulated differential genes were identified by PEG drought simulation induction during early germination of sensitive materials.Via KEGG enrichment analysis the 20 key drought resistance candidate genes were identified to be enriched in linoleic acid metabolism（csv00591）, pentose, glucuronic acid conversion（csv00040）, phenylpropanoid biosynthesis（csv00940）and plant hormone signal transduction（csv04075）pathways. The drought-resistant gene CsaV3_4G023820, CsaV3_2G006420 and two related SNP molecular markers were selected by associated SNP mutation site and RT-qPCR analysis.

Key words: cucumber, drought, transcriptome, molecular markers

谢洋, 周国彦, 苏航, 邢雨蒙, 闫立英. PEG模拟干旱条件下黄瓜种子发芽前后转录组分析[J]. 生物技术通报, 2023, 39(12): 148-157.

XIE Yang, ZHOU Guo-yan, SU Hang, XING Yu-meng, YAN Li-ying. Transcriptome Analysis of Cucumber Seeds Early and Late Germination Under PEG Drought Simulation[J]. Biotechnology Bulletin, 2023, 39(12): 148-157.

图/表 11

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样本 Sample	原始reads Raw reads	干净reads Clean reads	错误率 Error rate	Q20/%	GC比率 GC pct/%
DT1_CK	44 400 920	43 834 726	0.03	97.20	44.66
DT1_T	39 592 606	39 021 166	0.03	97.58	44.43
DS1_CK	41 022 958	40 686 226	0.03	97.10	44.02
DS1_T	42 691 442	42 269 126	0.03	97.30	44.04
DT2_CK	41 340 818	40 790 806	0.03	97.13	43.72
DT2_T	44 591 656	43 820 724	0.03	97.23	43.71
DS2_CK	42 263 290	41 550 102	0.03	97.34	43.66
DS2_T	43 966 094	43 058 866	0.03	97.22	43.81

样本 Sample	原始reads Raw reads	干净reads Clean reads	错误率 Error rate	Q20/%	GC比率 GC pct/%
DT1_CK	44 400 920	43 834 726	0.03	97.20	44.66
DT1_T	39 592 606	39 021 166	0.03	97.58	44.43
DS1_CK	41 022 958	40 686 226	0.03	97.10	44.02
DS1_T	42 691 442	42 269 126	0.03	97.30	44.04
DT2_CK	41 340 818	40 790 806	0.03	97.13	43.72
DT2_T	44 591 656	43 820 724	0.03	97.23	43.71
DS2_CK	42 263 290	41 550 102	0.03	97.34	43.66
DS2_T	43 966 094	43 058 866	0.03	97.22	43.81

KEGG ID	基因ID Gene ID	RPKM值 RPKM value						基因描述 Gene description
KEGG ID	基因ID Gene ID	DT1_T	DT1_CK	log₂FC	DS1_T	DS1_CK	log₂FC	基因描述 Gene description
csv00591	CsaV3_4G023820	2 660.909	177.544	3.905	8 268.943	3.90E+02	4.406	亚油酸13S -脂氧合酶2-1 Linoleate 13S-lipoxygenase 2-1
	CsaV3_2G006420	20.813	1.39E-17	7.391	2 200.098	7.36E+01	4.899	亚油酸9S -脂氧合酶4 Linoleate 9S-lipoxygenase 4
	CsaV3_4G023810	9.458	1.39E-17	6.263	282.605	46.658	2.595	亚油酸13S -脂氧合酶2-1 Linoleate 13S-lipoxygenase 2-1
csv00040	CsaV3_2G025090	223.315	7.402	4.893	1 319.479	129.599	3.347	果胶裂解酶5 Pectate lyase 5
	CsaV3_3G011280	101.247	9.516	3.395	670.348	58.062	3.527	果胶裂解酶8 Pectate lyase 8
	CsaV3_3G028700	459.883	80.320	2.516	1 844.185	193.879	3.249	果胶裂解酶8 Pectate lyase 8
	CsaV3_2G014800	34.061	7.402	2.184	506.377	1.24E+01	5.333	果胶甲基酯酶11 Pectin methylesterase 11, PME11
csv00940	CsaV3_1G042480	4329.184	54.957	6.296	34 647.010	1 795.690	4.270	β-葡糖苷酶12 Beta-glucosidase 12
	CsaV3_6G006890	294.286	26.424	3.471	1 065.807	1.39E-17	13.059	过氧化物酶61 Peroxidase 61
	CsaV3_3G030410	80.427	19.027	2.073	217.017	31.106	2.798	4-香豆酸-辅酶a连接酶异构体 4-coumarate--CoA ligase isoform 7,4CL7
	CsaV3_2G035150	14.190	1.39E-17	6.842	658.773	9.334	6.122	氧化物酶39 Peroxidase 39,ATP19a
	CsaV3_2G009070	36.900	7.402	2.299	54.975	3.113	4.090	细胞色素P450 Cytochrome P450,CYP73A100
	CsaV3_6G039690	17.028	2.118	2.938	42.436	4.150	3.317	苯丙氨酸氨裂合酶 Phenylalanine ammonia-lyase
csv04075	CsaV3_2G013210	45.416	1.39E-17	8.511	2 138.368	17.628	6.913	激素响应蛋白 Auxin-responsive protein, IAA14
	CsaV3_5G023170	65.288	5.289	3.596	652.022	81.907	2.991	生长素转运蛋白3 Auxin transporter-like protein 3
	CsaV3_6G007970	7.566	4.86E+01	-2.663	87.770	5.187	4.049	激素响应蛋白 Auxin responsive protein
	CsaV3_7G027610	423.925	67.639	2.646	548.817	99.533	2.462	吲哚-3-乙酸诱导蛋白 Indole-3-acetic acid-induced protein, ARG13
	CsaV3_1G030250	20.813	1.062	4.148	64.621	5.187	3.608	EIN3结合F-box蛋白 EIN3-binding F-box protein 2
	CsaV3_2G004130	719.161	75.036	3.259	1 417.861	305.850	2.212	吲哚-3-乙酸诱导蛋白 Indole-3-acetic acid-induced protein, ARG13
	CsaV3_3G005590	17.975	1.39E-17	7.180	42.436	2.0767	4.275	吲哚-3-乙酸-氨基合成酶Indole-3-acetic acid-amido synthetase, GH3.6

KEGG ID	基因ID Gene ID	RPKM值 RPKM value						基因描述 Gene description
KEGG ID	基因ID Gene ID	DT1_T	DT1_CK	log₂FC	DS1_T	DS1_CK	log₂FC	基因描述 Gene description
csv00591	CsaV3_4G023820	2 660.909	177.544	3.905	8 268.943	3.90E+02	4.406	亚油酸13S -脂氧合酶2-1 Linoleate 13S-lipoxygenase 2-1
	CsaV3_2G006420	20.813	1.39E-17	7.391	2 200.098	7.36E+01	4.899	亚油酸9S -脂氧合酶4 Linoleate 9S-lipoxygenase 4
	CsaV3_4G023810	9.458	1.39E-17	6.263	282.605	46.658	2.595	亚油酸13S -脂氧合酶2-1 Linoleate 13S-lipoxygenase 2-1
csv00040	CsaV3_2G025090	223.315	7.402	4.893	1 319.479	129.599	3.347	果胶裂解酶5 Pectate lyase 5
	CsaV3_3G011280	101.247	9.516	3.395	670.348	58.062	3.527	果胶裂解酶8 Pectate lyase 8
	CsaV3_3G028700	459.883	80.320	2.516	1 844.185	193.879	3.249	果胶裂解酶8 Pectate lyase 8
	CsaV3_2G014800	34.061	7.402	2.184	506.377	1.24E+01	5.333	果胶甲基酯酶11 Pectin methylesterase 11, PME11
csv00940	CsaV3_1G042480	4329.184	54.957	6.296	34 647.010	1 795.690	4.270	β-葡糖苷酶12 Beta-glucosidase 12
	CsaV3_6G006890	294.286	26.424	3.471	1 065.807	1.39E-17	13.059	过氧化物酶61 Peroxidase 61
	CsaV3_3G030410	80.427	19.027	2.073	217.017	31.106	2.798	4-香豆酸-辅酶a连接酶异构体 4-coumarate--CoA ligase isoform 7,4CL7
	CsaV3_2G035150	14.190	1.39E-17	6.842	658.773	9.334	6.122	氧化物酶39 Peroxidase 39,ATP19a
	CsaV3_2G009070	36.900	7.402	2.299	54.975	3.113	4.090	细胞色素P450 Cytochrome P450,CYP73A100
	CsaV3_6G039690	17.028	2.118	2.938	42.436	4.150	3.317	苯丙氨酸氨裂合酶 Phenylalanine ammonia-lyase
csv04075	CsaV3_2G013210	45.416	1.39E-17	8.511	2 138.368	17.628	6.913	激素响应蛋白 Auxin-responsive protein, IAA14
	CsaV3_5G023170	65.288	5.289	3.596	652.022	81.907	2.991	生长素转运蛋白3 Auxin transporter-like protein 3
	CsaV3_6G007970	7.566	4.86E+01	-2.663	87.770	5.187	4.049	激素响应蛋白 Auxin responsive protein
	CsaV3_7G027610	423.925	67.639	2.646	548.817	99.533	2.462	吲哚-3-乙酸诱导蛋白 Indole-3-acetic acid-induced protein, ARG13
	CsaV3_1G030250	20.813	1.062	4.148	64.621	5.187	3.608	EIN3结合F-box蛋白 EIN3-binding F-box protein 2
	CsaV3_2G004130	719.161	75.036	3.259	1 417.861	305.850	2.212	吲哚-3-乙酸诱导蛋白 Indole-3-acetic acid-induced protein, ARG13
	CsaV3_3G005590	17.975	1.39E-17	7.180	42.436	2.0767	4.275	吲哚-3-乙酸-氨基合成酶Indole-3-acetic acid-amido synthetase, GH3.6

基因ID Gene ID	数据库 Database	染色体CHROM	位置POS/bp	参考碱基EF	实际碱基ALT	质量QUAL	测序深度DP	基因型GT
CsaV3_4G023820	DS1-CK	chr.4	13 866 362	C	G	30.60	11	0/1
CsaV3_4G023820	DS1-T	chr.4	13 866 362	C	G	4 177.60	234	0/1
CsaV3_2G006420	DS1-CK	chr.2	3 052 439	C	T	254.60	11	0/1
CsaV3_2G006420	DS1-T	chr.2	3 052 439	C	T	852.60	194	0/1
CsaV3_2G025090	DT1-CK	chr.2	17 299 201	G	A	353.02	10	0/1
	DT1-T	chr.2	17 301 308	A	T	6 787.03	176	0/1
		chr.2	17 303 282	G	C	879.03	24	0/1
		chr.2	17 305 064	T	C	347.00	12	0/1
		chr.2	17 305 060	T	C	347.00	12	0/1