萱草根茎低温胁迫转录组分析

doi:10.3969/j.issn.1004-1524.2021.08.12

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (8): 1445-1460.DOI: 10.3969/j.issn.1004-1524.2021.08.12

萱草根茎低温胁迫转录组分析

黄长兵¹^,²(), 程培蕾², 杨绍宗³, 张焕朝¹^,^*(), 姜正之³, 金立敏²

1.南京林业大学南方现代林业协同创新中心/林学院,江苏南京 210037
2.苏州农业职业技术学院园艺科技学院,江苏苏州 215008
3.苏州市华冠园创园艺科技有限公司,江苏苏州 215557

收稿日期:2020-10-11 出版日期:2021-08-25 发布日期:2021-08-27
作者简介:*张焕朝,E-mail: hczhang@njfu.edu.cn
黄长兵(1979—),男,安徽六安人,硕士,副研究员,从事萱草抗寒性品种选育与冷胁迫下miRNA表达研究。E-mail: cbhuang@szai.edu.cn
通讯作者: 张焕朝
基金资助:
江苏省高等学校自然科学研究(19KJB210017);苏州市科技计划(SNG2018053);江苏现代农业产业技术体系(JATS〔2018〕232)

Transcriptome analysis of Hemerocallis fulva under low temperature stress

HUANG Changbing¹^,²(), CHENG Peilei², YANG Shaozong³, ZHANG Huanchao¹^,^*(), JIANG Zhengzhi³, JIN Limin²

1. Co-Innovation Center for the Sustainable Forestry in Southern China/ College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2. College of Horticulture, Suzhou Polytechnic Institute of Agriculture, Suzhou 215008, China
3. Suzhou Huaguan Garden Creative Horticulture Technology Co., Ltd., 215557, China

Received:2020-10-11 Online:2021-08-25 Published:2021-08-27
Contact: ZHANG Huanchao

摘要/Abstract

摘要：

低温胁迫是萱草育种和生产中最严重的非生物胁迫之一。该研究以耐冷型和冷敏感型萱草低温处理前后的根茎为材料,利用转录组测序(RNA-seq)技术,比较了耐冷型和冷敏感型萱草在低温胁迫下的基因表达差异。结果表明,4个样品获得的高质量clean reads均大于35 880 734条;低温处理后,耐冷型萱草差异表达基因数量多于冷敏感型,差异表达基因主要富集于次级代谢产物等相关途径,Ca²⁺信号通路基因、MAPKs信号通路基因、转录因子和热激蛋白基因表达在2种萱草中表现出不同的变化,可能导致2种萱草在低温胁迫中的不同反应。qRT-PCR验证结果显示,差异基因的表达变化趋势与RNA-seq结果一致。该研究表明Ca²⁺信号通路基因、MAPKs信号通路基因在萱草响应低温胁迫过程中发挥着重要作用。

关键词: 萱草, 转录组, 差异表达基因, 低温胁迫

Abstract:

Low temperature stress is one of the most serious abiotic stresses in Hemerocallis fulva breeding and production. In this study, the rhizomes of cold-tolerant and cold-sensitive H. fulva before and after low temperature treatment were used as materials, and the gene expression differences of cold-tolerant and cold-sensitive H. fulva under low temperature stress were compared using transcriptome sequencing (RNA-seq) technology. The results were as follows: More than 35 880 734 high-quality clean reads were obtained from all four samples; After low temperature treatment, the number of differentially expressed genes (DEGs) of cold-tolerant hemerocallis was higher than that of cold sensitive type, and the DEGs were mainly concentrated in secondary metabolites and other related pathways; The expression trends of Ca²⁺ signaling pathway genes, MAPKs signaling pathway genes, transcription factors, and heat shock protein genes showed different changes in the two Hemerocallis species, which might lead to different responses to low temperature stress. qRT-PCR results showed that the trends of DEGs expression were consistent with those of RNA-seq. These results indicated that Ca²⁺ signaling pathway genes and MAPKs signaling pathway genes played important role in H. fulva response to low temperature stress.

Key words: Hemerocallis fulva, transcriptome, differentially expressed genes, low temperature stress

中图分类号:

S682.1⁺9

黄长兵, 程培蕾, 杨绍宗, 张焕朝, 姜正之, 金立敏. 萱草根茎低温胁迫转录组分析[J]. 浙江农业学报, 2021, 33(8): 1445-1460.

HUANG Changbing, CHENG Peilei, YANG Shaozong, ZHANG Huanchao, JIANG Zhengzhi, JIN Limin. Transcriptome analysis of Hemerocallis fulva under low temperature stress[J]. Acta Agriculturae Zhejiangensis, 2021, 33(8): 1445-1460.

图/表 16

参考文献 41

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引物名称 Primer name	引物序列(5'-3') Sequence (5'-3')	基因 Gene	产物长度 Product length/bp
Unigene25337_All-F	CGTCCGGAGCCACAGCA	类硫氧还蛋白基因Thioredoxin-like protein gene	120
Unigene25337_All-R	AGAGGCAGCGAACCAG	类硫氧还蛋白基因Thioredoxin-like protein gene	120
Unigene25480_All-F	ATTGATGCTGCAAAAGGC	丝氨酸/精氨酸重复基质蛋白基因	118
		Aerine/arginine repetitive matrix protein 3-like gene
Unigene25480_All-R	TCTGTGTGTTTTCATAAA	丝氨酸/精氨酸重复基质蛋白基因	118
		Serine/arginine repetitive matrix protein 3-like gene
Unigene25572_All-F	TACTTTTCAGCTATTTAG	溶磷酰胆碱酰转移酶1基因	110
		Lysophos phatidylcholine acyltransferase 1 gene
Unigene25572_All-R	ACCCGGATGAAGTTTGC	溶磷酰胆碱酰转移酶1基因	110
		Lysophos phatidylcholine acyltransferase 1 gene
Unigene25602_All-F	GACGGCTGAGAGTGATGA	1-磷脂酰肌醇3-磷酸5-激酶基因	108
		1-phosphatidylinositol 3-phosphate 5-kinase gene
Unigene25602_All-R	ACAAGAAGAGGGAAAGG	1-磷脂酰肌醇3-磷酸5-激酶基因	108
		1-phosphatidylinositol 3-phosphate 5-kinase gene
Unigene25633_All-F	TCATCTTCTTCCTCTCTCTC	粘液素-2基因 Mucin-2 gene	110
Unigene25633_All-R	GCTTCTTCAGCGAAAGCTT	粘液素-2基因Mucin-2 gene	110
Unigene25700_All-F	TAAAACATCTATGTATTA	肌球蛋白VIIa基因 Myosin-VIIa-like gene	112
Unigene25700_All-R	TGTCGGGCAAACAGCTC	肌球蛋白VIIa基因 Myosin-VIIa-like gene	112
Unigene25732_All-F	GTCTGAATATTTCGTGT	含有LOB结构域的蛋白38基因	108
		LOB domain-containing protein 38-like gene
Unigene25732_All-R	CTGAATCTATGAGAATC	含有LOB结构域的蛋白38基因	108
		LOB domain-containing protein 38-like gene
Unigene25769_All-F	CAAGCTATTAATTTAAG	多聚泛素-A基因Polyubiquitin-A gene	106
Unigene25769_All-R	TTAGAGAAATCATGTCA	多聚泛素-A基因Polyubiquitin-A gene	106

引物名称 Primer name	引物序列(5'-3') Sequence (5'-3')	基因 Gene	产物长度 Product length/bp
Unigene25337_All-F	CGTCCGGAGCCACAGCA	类硫氧还蛋白基因Thioredoxin-like protein gene	120
Unigene25337_All-R	AGAGGCAGCGAACCAG	类硫氧还蛋白基因Thioredoxin-like protein gene	120
Unigene25480_All-F	ATTGATGCTGCAAAAGGC	丝氨酸/精氨酸重复基质蛋白基因	118
		Aerine/arginine repetitive matrix protein 3-like gene
Unigene25480_All-R	TCTGTGTGTTTTCATAAA	丝氨酸/精氨酸重复基质蛋白基因	118
		Serine/arginine repetitive matrix protein 3-like gene
Unigene25572_All-F	TACTTTTCAGCTATTTAG	溶磷酰胆碱酰转移酶1基因	110
		Lysophos phatidylcholine acyltransferase 1 gene
Unigene25572_All-R	ACCCGGATGAAGTTTGC	溶磷酰胆碱酰转移酶1基因	110
		Lysophos phatidylcholine acyltransferase 1 gene
Unigene25602_All-F	GACGGCTGAGAGTGATGA	1-磷脂酰肌醇3-磷酸5-激酶基因	108
		1-phosphatidylinositol 3-phosphate 5-kinase gene
Unigene25602_All-R	ACAAGAAGAGGGAAAGG	1-磷脂酰肌醇3-磷酸5-激酶基因	108
		1-phosphatidylinositol 3-phosphate 5-kinase gene
Unigene25633_All-F	TCATCTTCTTCCTCTCTCTC	粘液素-2基因 Mucin-2 gene	110
Unigene25633_All-R	GCTTCTTCAGCGAAAGCTT	粘液素-2基因Mucin-2 gene	110
Unigene25700_All-F	TAAAACATCTATGTATTA	肌球蛋白VIIa基因 Myosin-VIIa-like gene	112
Unigene25700_All-R	TGTCGGGCAAACAGCTC	肌球蛋白VIIa基因 Myosin-VIIa-like gene	112
Unigene25732_All-F	GTCTGAATATTTCGTGT	含有LOB结构域的蛋白38基因	108
		LOB domain-containing protein 38-like gene
Unigene25732_All-R	CTGAATCTATGAGAATC	含有LOB结构域的蛋白38基因	108
		LOB domain-containing protein 38-like gene
Unigene25769_All-F	CAAGCTATTAATTTAAG	多聚泛素-A基因Polyubiquitin-A gene	106
Unigene25769_All-R	TTAGAGAAATCATGTCA	多聚泛素-A基因Polyubiquitin-A gene	106

样品 Sample	原始 readsTotal raw reads	过滤后的reads Total clean reads	核苷酸数量 Number of nucleotides	质量值≥20碱基所占百分比 Q20/%	质量值≥30的碱基所占百分比 Q30/%	GC含量 GCcontent/%
4CK	47 738 570	46 171 660	6 858 685 119	98.61	95.65	48.83
4T	50 110 130	47 791 962	7 099 131 160	98.63	95.70	48.89
7CK	50 918 552	47 411 290	7 047 333 003	98.72	95.97	48.57
7T	38 540 808	35 880 734	5 342 671 114	98.82	96.23	48.48

样品 Sample	原始 readsTotal raw reads	过滤后的reads Total clean reads	核苷酸数量 Number of nucleotides	质量值≥20碱基所占百分比 Q20/%	质量值≥30的碱基所占百分比 Q30/%	GC含量 GCcontent/%
4CK	47 738 570	46 171 660	6 858 685 119	98.61	95.65	48.83
4T	50 110 130	47 791 962	7 099 131 160	98.63	95.70	48.89
7CK	50 918 552	47 411 290	7 047 333 003	98.72	95.97	48.57
7T	38 540 808	35 880 734	5 342 671 114	98.82	96.23	48.48

数据库 Database	数量 Number	比例 Percentage/%
NCBI非冗余蛋白数据库 Nr	38 447	44.15
NCBI核酸数据库 Nt	10 453	12.00
瑞士蛋白序列数据库 SwissProt	28 095	32.27
京都基因和基因组路径 KEGG	27 835	31.97
真核生物直系同源物 KOG	41 418	47.57
Pfam蛋白家族数据库 Pfam	36 767	42.23
基因本体GO	15 933	18.30

萱草根茎低温胁迫转录组分析

Transcriptome analysis of Hemerocallis fulva under low temperature stress

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参考文献 41

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路径 Pathway	代谢通路 Metabolic pathway	基因数量 Gene number	P值 P value
ko01100	异黄酮生物合成Isoflavone biosynthesis	5 543	1.23×10^-2
ko05010	牛磺酸和亚牛磺酸代谢Taurine and taurine metabolism	2 333	1.00×10^-2
ko04022	光合天线作用蛋白Photosynthetic antenna acting protein	2 134	8.15×10^-2
ko04022	二苯基庚酮和姜酚生物合成Biosynthesis of diphenylheptanone and gingerol	2 057	1.71×10^-3
ko04020	昼夜节律-植物Circadian rhythm-plants	2 026	3.72×10^-2
ko05169	不饱和脂肪酸的生物合成Biosynthesis of unsaturated fatty acids	1 915	1.00×10^-2
ko04972	花青素生物合成Anthocyanin biosynthesis	1 743	1.00×10^-2
ko05016	类黄酮生物合成Flavonoid biosynthesis	1 599	3.45×10^-3
ko00230	吲哚生物碱生物合成Biosynthesis of indole alkaloids	1 488	1.00×10^-2
ko05168	类固醇生物合成Steroid biosynthesis	1 447	1.00×10^-2
ko05110	嘌呤代谢purine metabolism	1 429	4.34×10^-3
ko00240	硫辛酸代谢Lipoic acid metabolism	1 345	1.00×10^-2
ko05146	黄酮生物合成Flavone biosynthesis	1 265	4.53×10^-2
ko03020	花生四烯酸代谢Arachidonic acid metabolism	1 121	9.80×10^-9
ko03010	RNA聚合酶RNA polymerase	1 110	6.73×10^-15
ko05152	光合作用Photosynthesis	990	1.00×10^-4

比较 Comparison	基因ID GeneID	log₂(fold change)	错误发现率 FDR	P值 P value	描述 Description
4CK-VS-4T	Unigene26840_All	1.752 123	7.08×10^-12	6.43×10^-13	CBL互作丝氨酸/苏氨酸蛋白激酶3
					CBL-interacting serine/threonine-protein kinase 3
7CK-VS-7T	CL2505.Contig6_All	1.261 008	1.64×10^-6	4.186×10^-7	钙/钙调蛋白依赖性蛋白激酶II
					Calcium/calmodulin-dependent protein kinase II alpha (CAMK2A)
	CL11891.Contig1_All	1.245 642	0	0	钙调蛋白Calmodulin (Calm5)
	CL10570.Contig1_All	1.240 229	0	0	钙调蛋白Calmodulin (Calm3)
	CL7725.Contig5_All	-2.088 160	2.18×10^-17	3.17×10^-18	钙调素和肌素相互作用RhoGEF
					Calmodulin and titin-interacting RhoGEF
	CL2721.Contig3_All	-2.334 250	1.38×10^-47	9.96×10^-49	CBL互作丝氨酸/苏氨酸蛋白激酶14
					CBL-interacting serine/threonine-protein kinase 14-like
4CK-VS-7CK	Unigene66_All	3.843 314	5.71×10^-111	2.74×10^-112	钙调蛋白Calmodulin (Calm1)
	CL11891.Contig3_All	1.867 317	0	0	钙调蛋白Calmodulin (Calm5)
	CL2721.Contig2_All	2.743 697	0	0	CBL互作丝氨酸/苏氨酸蛋白激酶14
					CBL-interacting serine/threonine-protein kinase 14-like
	CL2505.Contig6_All	-2.933 600	6.70×10^-48	6.57×10^-49	钙/钙调蛋白依赖性蛋白激酶II
					Calcium/calmodulin-dependent protein kinase II alpha (CAMK2A)
4T-VS-7T	Unigene66_All	3.345 624	1.17×10^-70	6.59×10^-72	钙调蛋白Calmodulin (Calm1)
	CL11891.Contig3_All	1.990 892	0	0	钙调蛋白Calmodulin (Calm5)
	CL2505.Contig6_All	-2.505 860	4.20×10^-58	2.86×10^-59	钙/钙调蛋白依赖性蛋白激酶II
					Calcium/calmodulin-dependent protein kinase II alpha (CAMK2A)
	CL7725.Contig5_All	-1.397 450	1.19×10^-6	3.68×10^-7	钙调素和肌素相互作用RhoGEF
					Calmodulin and titin-interacting RhoGEF
	Unigene6945_All	-1.307 820	1.57×10^-27	2.01×10^-28	钙调素结合转录激活子3
					Calmodulin-binding transcription activator 3

比较 Comparison	基因ID Gene ID	log₂(fold change)	错误发现率 FDR	P值 P value	描述 Description
4CK-VS-4T	CL1729.Contig2_All	1.968 445	9.05×10^-12	8.28×10^-13	丝裂原活化蛋白激酶激酶激酶3
					Mitogen-activated protein kinase kinase kinase kinase 3
	CL4304.Contig1_All	-1.360 070	6.30×10^-25	2.99×10^-26	丝裂原活化蛋白激酶1
					Mitogen-activated protein kinase 1
	CL5093.Contig4_All	-1.116 620	7.50×10^-11	7.33×10^-12	丝裂原活化蛋白激酶激酶激酶5
					Mitogen-activated protein kinase kinase kinase kinase 5
7CK-VS-7T	CL3036.Contig2_All	3.523 562	0.008 842 093	0.00 348 524	丝裂原活化蛋白激酶激酶激酶MLK4
					Mitogen-activated protein kinase kinase kinase MLK4-like
	CL1729.Contig2_All	2.092 002	2.45×10^-27	2.64×10^-28	丝裂原活化蛋白激酶激酶激酶3
					Mitogen-activated protein kinase kinase kinase kinase 3
	CL5625.Contig1_All	1.376 741	1.02×10^-18	1.42×10^-19	丝裂原活化蛋白激酶4
					Mitogen-activated protein kinase 4
	CL4304.Contig1_All	-3.903 960	3.62×10^-103	1.43×10^-104	丝裂原活化蛋白激酶1
					Mitogen-activated protein kinase 1
	CL12049.Contig2_All	-3.286 680	5.31×10^-18	7.56×10^-19	丝裂原激活蛋白激酶7
					Mitogen-activated protein kinase 7-like
	CL5093.Contig4_All	-1.990 600	1.35×10^-10	2.63×10^-11	丝裂原活化蛋白激酶激酶激酶 5
					Mitogen-activated protein kinase kinase kinase kinase 5
4CK-VS-7CK	CL8048.Contig2_All	3.014 950	1.08×10^-88	6.41×10^-90	丝裂原激活蛋白激酶8互作蛋白2
					Mitogen-activated protein kinase 8 interacting protein 2 (MAPK8IP2)
	CL1729.Contig2_All	1.106 915	0.001 163 593	0.000 507 178	丝裂原激活蛋白激酶激酶激酶3
					Mitogen-activated protein kinase kinase kinase kinase 3
	CL12049.Contig2_All	1.062 509	1.48×10^-5	5.34×10^-6	丝裂原激活蛋白激酶7
					Mitogen-activated protein kinase 7-like
	CL5625.Contig1_All	-1.516 540	5.41×10^-25	8.56×10^-26	丝裂原活化蛋白激酶4
					Mitogen-activated protein kinase 4
	CL5093.Contig4_All	-1.044 110	4.97×10^-10	1.34×10^-10	丝裂原活化蛋白激酶激酶激酶 5
					Mitogen-activated protein kinase kinase kinase kinase 5
4T-VS-7T	CL8048.Contig2_All	4.202 332	1.56×10^-200	3.03×10^-202	丝裂原激活蛋白激酶8互作蛋白2
					Mitogen-activated protein kinase 8 interacting protein 2 (MAPK8IP2)
	CL1729.Contig2_All	1.230 472	1.80×10^-13	3.76×10^-14	丝裂原激活蛋白激酶激酶激酶3
					Mitogen-activated protein kinase kinase kinase kinase 3
	CL3036.Contig2_All	-2.181 490	8.88×10^-6	2.93×10^-6	丝裂原活化蛋白激酶激酶激酶 MLK4
					Mitogen-activated protein kinase kinase kinase MLK4-like
	CL12049.Contig2_All	-2.147 640	8.88×10^-6	2.93×10^-6	丝裂原激活蛋白激酶7
					Mitogen-activated protein kinase 7-like
	CL4304.Contig1_All	-2.051 190	4.72×10^-15	9.22×10^-16	丝裂原活化蛋白激酶1
					Mitogen-activated protein kinase 1
	CL5093.Contig4_All	-1.918 090	1.10×10^-9	2.80×10^-10	丝裂原活化蛋白激酶激酶激酶 5
					Mitogen-activated protein kinase kinase kinase kinase 5

比较 Comparison	基因ID Gene ID	log₂ fold change	错误发现率 FDR	P值 P value	描述 Description
4CK-VS-4T	Unigene9010_All	1.953 179	1.12×10^-221	5.96×10^-224	HSP70-2
	CL4189.Contig1_All	1.291 709	4.47×10^-12	4.00×10^-13	DnaJ热激蛋白
					DnaJ heat shock protein (DNAJC6)
	Unigene6560_All	1.045 864	1.83×10^-25	8.58×10^-27	HSP90
7CK-VS-7T	Unigene14974_All	1.272 815	4.91×10^-21	6.36×10^-22	HSP86
	Unigene6560_All	-1.611 640	8.90×10^-22	1.12×10^-22	HSP90
	CL2382.Contig6_All	-1.455 820	3.29×10^-8	7.40×10^-9	HSP90-5
4CK-VS-7CK	CL12755.Contig2_All	3.497 080	1.25×10^-231	2.95×10^-233	HSPA8
	CL2382.Contig6_All	-4.260 570	0	0	HSP90-5
	CL5229.Contig2_All	-1.364 170	1.90×10^-45	1.95×10^-46	HSP70-2
4T-VS-7T	CL2382.Contig6_All	-4.985 870	1.10×10^-307	0	HSP90-5
	Unigene6560_All	-2.617 300	6.68×10^-79	3.36×10^-80	HSP90
	Unigene9010_All	-1.729 100	2.73×10^-161	6.80×10^-163	HSPA8
	CL707.Contig3_All	-1.140 870	2.44×10^-220	4.23×10^-222	HSP60

比较 Comparison	基因ID Gene ID	log₂ fold change	错误发现率 FDR	P值 P value	描述 Description
4CK-VS-4T	CL703.Contig1_All	-1.440 740	1.94×10^-14	1.50×10^-15	WRKY11
	Unigene4802_All	-3.694 380	1.60×10^-80	2.43×10^-82	MYB102
7CK-VS-7T	CL4950.Contig5_All	2.192 645	4.83×10^-10	9.70×10^-11	MYB20
	CL9135.Contig2_All	1.171 953	7.09×10^-66	4.01×10^-67	bHLH2
	Unigene4802_All	-1.900 390	9.78×10^-32	9.5×10^-33	MYB102
4CK-VS-7CK	CL703.Contig1_All	2.076 462	2.29×10^-100	1.21×10^-101	WRKY11
	CL9135.Contig2_All	-2.614 170	0	0	bHLH2
	CL4950.Contig5_All	-1.890 000	3.43×10^-7	1.09×10^-7	MYB122-like
4T-VS-7T	CL703.Contig1_All	3.401 090	2.74×10^-152	7.22×10^-154	WRKY11
	Unigene4802_All	1.723 331	1.95×10^-9	5.02×10^-10	MYB122-like
	CL9135.Contig2_All	-1.895 210	0	0	bHLH2

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