结合RNA-seq分析和QTL定位筛选甘蓝型油菜萌发期与铝毒胁迫相关的候选基因

doi:10.3724/SP.J.1006.2021.04231

Abstract

Abstract:

With the aggravation of soil acidification, aluminum toxicity has become one of the important stress factors affecting seed germination quality and crop yield. In order to study the molecular mechanism of the effect of aluminum toxicity on rapeseed seed germination, a total of 9344 significantly differentially expressed genes [log₂(fold change) ≥ 1 and FDR ≤ 0.05] were detected in the transcriptome analysis of aluminum-tolerant strain 18D300 and aluminum-sensitive strain 27011 by RNA-seq technology, among which 4406 DEGs (differentially expressed genes) were up-regulated and 4938 DEGs were down-regulated. GO enrichment showed that DEGs were mainly related to oxidation reaction, carbohydrate metabolism, and transporter activity. KEGG enrichment revealed that DEGs were mainly concentrated in phenylpropane biosynthesis, starch and sucrose metabolism, MAPK signal pathway-plant, plant-pathogen interaction, plant hormone signal transduction and so on. In addition, 44 DEGs (10 down-regulated and 34 up-regulated) were screened by integrating the results of RNA transcriptome sequencing and QTL mapping of root-related traits at germination stage under aluminum toxicity stress in rapeseed, which were mainly related to oxidative stress, osmotic regulation, cell wall modification, transporter, and hormone signal transduction.

Key words: Brassica napus L., aluminum poison, transcriptome sequencing, QTLs, differentially expressed genes

WANG Rui-Li, WANG Liu-Yan, LEI Wei, WU Jia-Yi, SHI Hong-Song, LI Chen-Yang, TANG Zhang-Lin, LI Jia-Na, ZHOU Qing-Yuan, CUI Cui. Screening candidate genes related to aluminum toxicity stress at germination stage via RNA-seq and QTL mapping in Brassica napus L.[J].Acta Agronomica Sinica, 2021, 47(12): 2407-2422.

Figures/Tables 15

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

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Table 3

KEGG pathway significantly overrepresented in R0 vs RT and S0 vs ST"

KEGG编号KEGG ID	KEGG术语 KEGG term		基因数目 Genes number		比值 Ratio (%)			矫正后的P值 P-value
R0 vs RT
bna00940	苯丙烷生物合成Phenylpropanoid biosynthesis		55		21.32			3.81844E-21
bna00500	淀粉和蔗糖代谢Starch and sucrose metabolism		27		10.47			2.29236E-06
bna00910	氮素代谢Nitrogen metabolism		13		5.04			1.81952E-05
bna00270	半胱氨酸和蛋氨酸代谢CySteine and methionine metabolism		21		8.14			0.000110181
bna04016	MAPK信号通路-植物MAPK signaling pathway-plant		23		8.91			0.000143365
bna00010	糖酵解/糖异生Glycolysis/Gluconeogenesis		21		8.14			0.000268893
bna00130	泛醌和其他萜类醌生物合成 Ubiquinone and other terpenoid-quinone biosynthesis		11		4.26			0.001478368
bna00380	色氨酸代谢Tryptophan metabolism		12		4.65			0.003486812
bna00360	苯丙氨酸代谢Phenylalanine metabolism		10		3.88			0.006641539
KEGG编号KEGG ID		KEGG术语 KEGG term		基因数目 Genes number		比值 Ratio (%)	矫正后的P值 P-value
bna00460		氰基氨基酸代谢Cyanoamino acid metabolism		12		4.65	0.006641539
bna00350		酪氨酸代谢Tyrosine metabolism		9		3.49	0.007588613
bna04075		植物激素信号转导Plant hormone signal transduction		34		13.18	0.007588613
bna00052		半乳糖代谢Galactose metabolism		10		3.88	0.009316627
S0 vs ST
bna00940		苯丙烷生物合成Phenylpropanoid biosynthesis		97		31.80	5.89634E-43
bna04016		MAPK信号通路-植物MAPK signaling pathway-plant		38		12.46	9.09024E-08
bna00910		氮素代谢Nitrogen metabolism		16		5.25	2.123E-05
bna00460		氰基氨基酸代谢Cyanoamino acid metabolism		19		6.23	0.000428636
bna04075		植物激素信号转导Plant hormone signal transduction		52		17.05	0.001727744
bna00520		氨基糖和核苷酸糖代谢 Amino sugar and nucleotide sugar metabolism		27		8.85	0.004361711
bna04626		植物-病原体相互作用Plant-pathogen interaction		35		11.48	0.004455032
bna00040		戊糖和葡糖醛酸相互转化 Pentose and glucuronate interconversions		21		6.89	0.005458802

Table 3

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Table 4

Candidate genes possibly involved in aluminum poison coercion using QTL and RNA-Seq techniques"

性状 Trait	位点 QTL	甘蓝型油菜基因编号 Gene ID in B. napus	基因登录号 Gene accession	调节 Regulated	RT:fpkm	ST:fpkm	基因注释 Gene annotations
相对发芽势RGV				Up	19.6	0.87
		BnaA01g31480D	AT3G10720	Up	58.3	16.8	植物转化酶/果胶甲基酯酶抑制剂超家族 Plant invertase/Pectin methyl esterase inhibitor superfamily
		BnaA01g30490D	AT3G12580	Up	176	56.5	热激蛋白70heatshockprotein70,hsp70
		BnaA01g28900D	AT3G15450	Up	264	30	Al具有YGL和LRDR图案的铝诱导蛋白质 Al-induced protein with YGL and LRDR patterns
		BnaA01g27670D	AT3G16690	Up	11.7	0.47	结蛋白MtN3家族蛋白Nodin MtN3 family protein
	qRGV-A03-1	BnaA03g50730D	AT3G48000	Down	0.08	13.5	乙醛脱氢酶2B4Acetaldehyde dehydrogenase 2B4
		BnaA03g50130D	AT4G30420	Up	20.9	4.05	结蛋白MtN21 /EamA样转运蛋白家族蛋白 Nodin MtN21 /EamA-like transporter family protein
		BnaA03g50050D	AT4G30270	Up	36.2	9.65	木葡聚糖内葡聚糖酰化酶/水解酶 (XTH) Xyloglucan endoglucanase/hydrolase (XTH)
		BnaA03g47720D	AT4G26010	Up	6.4	0.7	过氧化物酶超家族蛋白Peroxidase superfamily protein
		BnaA03g47250D	AT4G25260	Up	36.6	4.89	植物转化酶/果胶甲基酯酶抑制剂超家族 Plant invertase/Pectin methyl esterase inhibitor superfamily
		BnaA03g46060D	AT4G23590	Up	11	0.16	酪氨酸转氨酶家族蛋白Tyrosine transaminase family protein
		BnaA03g45950D	AT4G00430	Up	18.7	2.9	跨膜蛋白CTransmembrane protein c
		BnaA03g44820D	AT4G21680	Down	3.21	4.06	硝酸盐转运体Nitrate transporter
		BnaA03g43140D	AT4G17550	Up	10.3	0.05	主要促进因子超家族蛋白质Major promoter superfamily proteins
		BnaA03g42860D	AT4G16820	Up	6.25	0.62	α/β-水解酶超家族蛋白质α/β-hydrolase superfamily protein
		BnaA03g42310D	AT4G15960	Up	17.6	4.18	α/β-水解酶超家族蛋白质α/β-hydrolase superfamily protein
		BnaA03g40990D	AT3G50830	Down	0.31	17.7	冷调节413等离子体Cold conditioning 413 plasma
		BnaA03g40380D	AT5G61600	Up	173	31.6	乙烯响应因子104 (ERF104) Ethylene response factor 104 (ERF104)
	qRGV-A03-2	BnaA03g09460D	AT5G59530	Down	1.94	14.8	2-氧代戊二酸和铁依赖性加氧酶超家族蛋白 2-oxoglutarate and iron-dependent oxygenase superfamily proteins
		BnaA03g09400D	AT5G59550	Up	42.4	10.6	锌指(C3HC4型无名指)家族蛋白 Zinc finger (C3HC4 ring finger) family protein
		BnaA03g09290D	AT5G59730	Up	27.3	5.38	外胚层亚单位exo70家族蛋白 Exoderm subunit exo70 family protein
		BnaA03g08870D	AT5G60530	Up	10.7	0.26	晚期胚胎发生丰富蛋白/ LEA蛋白 Late embryogenesis rich protein/LEA protein
		BnaA03g08820D	AT5G60660	Up	44.5	0.84	质膜内在蛋白2 Plasma membrane intrinsic protein 2
		BnaA03g07790D	AT5G19855	Down	3.53	6.86	伴侣蛋白RbcX Companion protein RbcX
		BnaA03g07660D	AT5G19600	Down	2.83	21.8	硫酸盐转运蛋白Sulfate transporter
		BnaA03g03740D	AT5G12020	Up	99.8	18.3	17.6 kD二类热激蛋白(HSP17.6II) 17.6 kD class ii heat shock protein (HSP17.6II)
	qRGV-C03	BnaC03g08610D	AT5G17820	Up	14.7	0.78	过氧化物酶超家族蛋白Peroxidase superfamily protein
相对发芽率RGR	qRGR-C04	BnaC04g10740D	AT2G34500	Up	55.9	12.3	细胞色素P450,家族710,亚家族A,多肽1 (CYP710) Cytochrome P450, family 710, subfamily A, polypeptide 1 (CYP710)
		BnaC04g08480D	AT2G36780	Down	0.9	23.3	UDP-糖基转移酶超家族蛋白 UDP-glycosyltransferase superfamily protein
		BnaC04g07650D	AT2G37820	Up	10.1	0.22	富含半胱氨酸/组氨酸结构域家族蛋白 Protein rich in cysteine/histidine domain family
		BnaC04g06480D	AT2G38940	Down	3.11	182	磷酸盐转运蛋白Phosphate transporter
		BnaC04g06140D	AT2G39430	Up	33.4	5.44	抗病反应(定向蛋白样)家族蛋白 Disease resistance response (directed protein-like) family protein
相对根长RRL	qRRL-A03-2	BnaA03g53200D	AT4G35180	Down	0.46	0.31	赖氨酸/组氨酸转运蛋白7 (LHT7) Lysine/histidine transporter 7 (LHT7)
	qRRL-A03-2	BnaA03g52830D	AT4G34410	Up	17.2	2.6	氧化还原反应转录因子1 (RRTF1) Redox transcription factor 1 (RRTF1)
	qRRL-A09	BnaA09g02040D	AT4G12550	Up	37.1	0	生长素诱导的根培养Root culture induced by auxin
	qRRL-A09	BnaA09g02030D	AT4G12550	Up	470	34.5	生长素诱导的根培养1Auxin-induced root culture 1
	qRRL-C03	BnaC03g65590D	AT4G35060	Down	4.57	6.67	重金属转运/解毒超家族蛋白 Heavy metal transport/detoxification superfamily protein
		BnaC03g65200D	AT4G23550	Up	13.6	1.28	WRKY29家族WRKY29 familyWRKY29 family wrky 29 family
		BnaC03g62130D	AT4G23700	Up	256	70.6	阳离子/氢离子交换器17 (CHX17) Cation/hydrogen ion exchanger 17 (CHX17)
相对芽长RBL	qRBL-C08	BnaC08g17880D	AT1G18300	Up	115	28.1	nudix水解酶同源物(NUDT) Nudix hydrolase homologue (NUDT)
相对芽长RBL		BnaC08g16940D	AT1G16390	Up	1.89	0.05	有机阳离子/肉碱转运蛋白Organic cation/carnitine transporter
相对干重RDW	qRDW-A09-1	BnaA09g30120D	AT1G23090	Up	0.04	0.66	硫酸盐转运蛋白(AST) Sulfate transporter (AST)
	qRDW-A09-1	BnaA09g29940D	AT4G13390	Up	38.4	3.17	富含脯氨酸的伸展蛋白样家族蛋白 Proline-rich extension-like family proteins
	qRDW-A10-2	BnaA10g25970D	AT5G04250	Down	7.13	22.2	半胱氨酸蛋白酶超家族蛋白 Cysteine protease superfamily protein

Table 4

Fig. 9

Fig. 10

Table 5

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样品名称 Sample name	S0	R0	ST	RT	总数 Total
原始读数Raw reads	48,585,792	55,778,592	45,860,428	47,308,644	197,533,456
干净读数Clean reads	44,642,424	51,491,214	41,819,768	43,518,768	181,472,174
干净碱基Clean data (bp)	6,696,363,600	7,723,682,100	6,272,965,200	6,527,815,200	27,220,826,100
N (%)	0.001385	0.00138	0.001418	0.00137
Q20 (%)	97.57	97.59	97.72	97.43
Q30 (%)	94.14	94.22	94.43	93.74

基因名称 Gene name	正向引物 Forward primer (5°-3°)	反向引物 Reverse primer (5°-3°)
BraACTIN7	GGAGCTGAGAGATTCCGTTG	GAACCACCACTGAGGACGAT
BnaA03g47720D	ACAAAGCAATGCACTCTTTAGG	GTTGTTGAAGACTCTGCAGTTT
BnaC03g62130D	AACAAAGACAAACCTGGGACTA	CTTGTTGATCGAACGGTAATCG
BnaA09g02030D	CTTCTGCTCAAGGCTCT	GGGTTTCCCAATGTTAG
BnaA03g03740D	GAAGTATGTGAGGATGGAGAGG	GATCTTCTCCAAATCAGCGTTC
BnaA03g53200D	GCCATTGTTGTATATTGGGCAT	TGATGTGTATGAAACAAGCAGC
BnaC03g65590D	ACGTTGAGCCAAACAGAGTG	CTATTATCGTAGACACCGGACG

生理指标 Physiological index	R系 R strain			S系 S strain
生理指标 Physiological index	R0	RT	增幅 Range of increase (%)	S0	ST	增幅 Range of increase (%)
丙二醛 MDA (nmol g^-1)	4.26	4.44	0.04	5.63	6.16	0.10^**
苯丙氨酸解氨酶 PAL (U g^-1)	4.54	6.53	0.44	5.06	6.45	0.27
脯氨酸 Pro (µg g^-1)	51.26	56.70	0.11^**	52.39	57.20	0.09^**
超氧化物歧化酶 SOD (U g^-1)	54.89	86.50	0.58^**	58.63	66.13	0.13^**
过氧化氢酶 CAT (U g^-1)	281.71	261.39	-0.07^**	376.24	282.13	-0.25^**
过氧化物酶 POD (U g^-1)	14,521.79	10,566.33	-0.27	13,381.75	10,337.50	-0.23^**
可溶性糖 SUG (mg g^-1)	2.18	1.80	-0.17^**	3.12	2.85	-0.09^**

Screening candidate genes related to aluminum toxicity stress at germination stage via RNA-seq and QTL mapping in Brassica napus L.

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