基于RNA-Seq筛选高粱低氮胁迫相关候选基因

doi:10.3724/SP.J.1006.2024.34055

Abstract

Abstract:

The objective of this study is to explore gene differential expression between different sorghum materials under low nitrogen stress conditions and to provide the references for probing into the breeding of low-nitrogen-tolerant sorghum varieties and the molecular mechanism of low-nitrogen-stress tolerance in sorghum. Two low-nitrogen-tolerant sorghum varieties (BSX44 and BTx378) were selected as experimental materials, and both of them were subjected to normal-growth treatment and low-nitrogen-stress treatment respectively before the gene expression of sorghum was detected at seedling stage, heading stage and flowering stage via RNA-Seq technology. The biological functions and metabolic pathways of the differentially expressed genes (DEGs) were analyzed by bioinformatics to screen genes that may be involved in the low-nitrogen regulation, and to understand the possible molecular pathways for nitrogen efficient materials in the process of nitrogen absorption and utilization. The results showed that: For BTx378 and BSX44, under normal-growth and low-nitrogen-stress treatments, 937 and 787 DEGs were detected at the seedling stage, 1305 and 935 at the heading stage, and 1402 and 963 at the flowering stage, for BTx378 and BSX44 respectively. Then the converged DEGs at the three stages were identified, and it was found that 246 genes were differentially expressed in the two low-nitrogen-tolerant sorghum varieties at the seedling stage, 371 at the heading stage, and 306 at the flowering stage. Furthermore, a total of 28 genes were consistently detected as DEGs at all three stages in the two low-nitrogen tolerant varieties, among which 5 genes were up-regulated and 23 genes were down-regulated. The KEGG analysis of the 28 common DEGs showed that they were mainly enriched in nitrogen metabolism, alanine, aspartic acid and glutamic acid metabolism, glycerophospholipid metabolism, and amino acid biosynthesis. This suggested that regulation of the genes in these pathways mainly affects the low nitrogen stress tolerance in sorghum.

Key words: sorghum, transcriptome sequencing, low nitrogen stress, differentially expressed genes (DEGs)

WANG Rui, ZHANG Fu-Yao, ZHAN Peng-Jie, CHU Jian-Qiang, JIN Min-Shan, ZHAO Wei-Jun, CHENG Qing-Jun. Identification of candidate genes implicated in low-nitrogen-stress tolerance based on RNA-Seq in sorghum[J].Acta Agronomica Sinica, 2024, 50(3): 669-685.

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Related information of common differentially expressed genes"

序号	基因编号	基因功能注释
Number	Gene ID	Gene function annotation
1	Sb01g006100↓	铁氧还蛋白——NADP还原酶, 根同工酶, 叶绿体 Ferredoxin—NADP reductase, root isozyme, chloroplastic
2	Sb01g007010↓	PAC2家族 PAC2 family
3	Sb01g015190↑	异天冬酰胺肽酶/L-天冬酰胺酶1亚基β Isoaspartyl peptidase/L-asparaginase 1 subunit beta
4	Sb01g023750↓	丙氨酸转氨酶2 Alanine aminotransferase 2
5	Sb01g029470↓	硝酸盐转运蛋白1.1 POT家族 Nitrate transporter 1.1 POT family
6	Sb01g032250↑	二酰基甘油激酶1 Diacylglycerol kinase 1
7	Sb02g006060↓	抗病蛋白RPM1 Disease resistance protein RPM1
8	Sb02g042310↓	磷脂酶A1-Igamma2, 叶绿体 Phospholipase A1-Igamma2, chloroplastic
9	Sb03g012720↓	磷脂酶D p1 Phospholipase D p1
10	Sb03g036210↓	紫色酸性磷酸酶2 Purple acid phosphatase 2
11	Sb03g045170↑	蛋白SRG1 Protein SRG1
12	Sb04g005740↓	细胞色素P450 71D10 Cytochrome P450 71D10
13	Sb04g006250↓	氯通道样蛋白CLC-g Putative chloride channel-like protein CLC-g
14	Sb04g006740↓	枯草杆菌蛋白酶Subtilisin-like protease
15	Sb04g021010↓	甘油磷酸二酯磷酸二酯酶家族 Glycerophosphoryl diester phosphodiesterase family
16	Sb04g032280↓	分歧的CRAL/TRIO域 Divergent CRAL/TRIO domain
17	Sb04g034160↓	铁氧还蛋白——亚硝酸盐还原酶, 叶绿体 Ferredoxin—nitrite reductase, chloroplastic
18	Sb04g035040↓	20 kD伴侣蛋白, 叶绿体20 kD chaperonin, chloroplastic
19	Sb05g016820↓	精氨酸/丝氨酸蛋白45 Arginine/serine-rich protein 45
20	Sb06g016540↓	脱落胁迫成熟蛋白2 Abscisic stress-ripening protein 2
21	Sb06g025950↑	含SPX结构域的膜蛋白 SPX domain-containing membrane protein
22	Sb06g031460↓	谷氨酰胺合成酶, 叶绿体Glutamine synthetase, chloroplastic
23	Sb07g008300↓	紫色酸性磷酸酶15 Purple acid phosphatase 15
24	Sb07g026000↓	营养细胞壁蛋白gp1 Vegetative cell wall protein gp1
25	Sb07g028630↑	含BTB/POZ结构域的蛋白 BTB/POZ domain-containing protein
26	Sb08g003180↓	转录因子GLK2 Probable transcription factor GLK2
27	Sb09g028090↓	营养细胞壁蛋白gp1 Vegetative cell wall protein gp1
28	Sorghum_bicolor_newGene_166↓	未知蛋白Unknown protein

Table 6

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Fig. 8

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品种 Variety	生育期 Growth stage	处理Treatment
品种 Variety	生育期 Growth stage	正常氮Normal nitrogen	低氮Low nitrogen
BTx378	苗期 Seedling stage	T1 (NN-R1-S1)	T4 (LN-R1-S1)
	抽穗期 Heading stage	T2 (NN-R1-S2)	T5 (LN-R1-S2)
	开花期 Flowering stage	T3 (NN-R1-S3)	T6 (LN-R1-S3)
BSX44	苗期 Seedling stage	T7 (NN-R2-S1)	T10 (LN-R2-S1)
	抽穗期 Heading stage	T8 (NN-R2-S2)	T11 (LN-R2-S2)
	开花期 Flowering stage	T9 (NN-R2-S3)	T12 (LN-R2-S3)

对比组 Compare group		对照组vs处理组 Control group vs Treatment group
A	1	T1 vs T4	NN-R1-S1 vs LN-R1-S1	正常氮-BTx378-苗期 vs低氮-BTx378-苗期 Normal nitrogen-BTx378-Seedling stage vs Low nitrogen-BTx378-Seedling stage
A	2	T7 vs T10	NN-R2-S1 vs LN-R2-S1	正常氮-BSX44-苗期 vs低氮-BSX44-苗期 Normal nitrogen-BSX44-Seedling stage vs Low nitrogen-BSX44-Seedling stage
B	3	T2 vs T5	NN-R1-S2 vs LN-R1-S2	正常氮-BTx378-抽穗期 vs低氮-BTx378-抽穗期 Normal nitrogen-BTx378-Heading stage vs Low nitrogen-BTx378-Heading stage
B	4	T8 vs T11	NN-R2-S2 vs LN-R2-S2	正常氮-BSX44-抽穗期 vs低氮-BSX44-抽穗期 Normal nitrogen-BSX44-Heading stage vs Low nitrogen-BSX44-Heading stage
C	5	T3 vs T6	NN-R1-S3 vs LN-R1-S3	正常氮-BTx378-开花期 vs低氮-BTx378-开花期 Normal nitrogen-BTx378-Flowering stage vs Low nitrogen-BTx378-Flowering stage
C	6	T9 vs T12	NN-R2-S3 vs LN-R2-S3	正常氮-BSX44-开花期 vs低氮-BSX44-开花期 Normal nitrogen-BSX44-Flowering stage vs Low nitrogen-BSX44-Flowering stage

引物名称 Primer ID	序列 Sequence (5′-3′)	产物大小 Product length (bp)
Sb01g006100-F	TTGTCTCGGTGGAGAGGCTA	104
Sb01g006100-R	ATAACTCTGGCCCTCCCAGT	104
Sb01g015190-F	CTGATAATGGGTCTGGCGGT	157
Sb01g015190-R	CTTTTCCGAACGGCGACAAC	157
Sb01g029470-F	TCTGACCCGGACTTGCTCTA	107
Sb01g029470-R	TGGTCGAGGAACCTGCATTC	107
Sb04g032280-F	TGTGGGCGCACATAGACAAG	127
Sb04g032280-R	TCATACATGCAGCGGCTCTC	127
Sb06g016540-F	CGAAGGTGGCTACAACGAGT	171
Sb06g016540-R	TCGTTGGTGCCCGATTTGTT	171
Sb06g031460-F	ATGATGGGTCAAGCACAGGG	99
Sb06g031460-R	TGTTGTTGCCACCTCGGAAT	99
SbActin-F	ATTCACGAGACTACCTACAAC	84
SbActin-R	ACCAGAGAGGACGATGTT	84

百迈客样品编号 BMK-ID	样品 Sample	过滤序列 Clean reads	过滤碱基 Clean bases	GC 含量 GC content (%)	Q30 (%)
T1	NN-R1-S1	16,868,217	4,978,049,154	55.45	92.73
T2	NN-R1-S2	19,294,220	5,706,447,154	56.06	92.41
T3	NN-R1-S3	19,051,651	5,623,880,026	57.58	92.19
T4	LN-R1-S1	16,490,988	4,854,433,778	56.78	91.65
T5	LN-R1-S2	15,070,044	4,433,027,812	56.47	92.03
T6	LN-R1-S3	15,956,808	4,691,702,406	56.77	92.72
T7	NN-R2-S1	14,343,423	4,221,379,930	57.01	92.55
T8	NN-R2-S2	15,743,754	4,605,121,084	56.21	92.98
T9	NN-R2-S3	13,847,707	4,095,501,214	56.07	92.13
T10	LN-R2-S1	14,905,380	4,407,032,326	56.80	91.53
T11	LN-R2-S2	13,986,375	4,119,603,114	56.65	92.66
T12	LN-R2-S3	17,798,466	5,262,344,634	56.17	91.74

对比组 Compare group		比对组 Compare group	上调基因 Up-regulated genes	下调基因 Down-regulated genes	差异表达基因数目 DEG number
A	1	NN-R1-S1 vs LN-R1-S1	464	473	937
A	2	NN-R2-S1 vs LN-R2-S1	264	523	787
B	3	NN-R1-S2 vs LN-R1-S2	637	668	1305
B	4	NN-R2-S2 vs LN-R2-S2	432	503	935
C	5	NN-R1-S3 vs LN-R1-S3	394	1008	1402
C	6	NN-R2-S3 vs LN-R2-S3	384	579	963

Identification of candidate genes implicated in low-nitrogen-stress tolerance based on RNA-Seq in sorghum

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KO编号KO ID	KEGG通路 KEGG pathway	KEGG同源基因KEGG orthology	基因编号 Gene ID
ko00910	氮代谢Nitrogen metabolism	K00366+K01915	Sb04g034160; Sb06g031460
ko00250	丙氨酸、天冬氨酸和谷氨酸代谢 Alanine, aspartate and glutamate metabolism	K00814+K01915	Sb01g023750; Sb06g031460
ko00564	甘油磷脂代谢Glycerophospholipid metabolism	K00901+K01115	Sb01g032250; Sb03g012720
ko01230	氨基酸的生物合成Biosynthesis of amino acids	K00814+K01915	Sb01g023750; Sb06g031460
ko00565	醚脂代谢Ether lipid metabolism	K01115	Sb03g012720
ko01210	2-氧羧酸代谢2-Oxocarboxylic acid metabolism	K00814	Sb01g023750
ko00630	乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolism	K01915	Sb06g031460
ko04070	磷脂酰肌醇信号系统Phosphatidylinositol signaling system	K00901	Sb01g032250
ko00195	光合作用Photosynthesis	K02641	Sb01g006100
ko00561	甘油脂代谢Glycerolipid metabolism	K00901	Sb01g032250
ko00330	精氨酸和脯氨酸代谢Arginine and proline metabolism	K01915	Sb06g031460
ko00710	光合生物的碳固定 Carbon fixation in photosynthetic organisms	K00814	Sb01g023750
ko04144	内吞作用Endocytosis	K01115	Sb03g012720
ko01200	碳代谢Carbon metabolism	K00814	Sb01g023750

基因编号 Genome ID	功能 Function	同源基因 Orthologous genes		苗期 Seedling stage		抽穗期 Heading stage		开花期 Flowering stage
基因编号 Genome ID	功能 Function	玉米 Maize	水稻 Rice	T1 vs T4	T7 vs T10	T2 vs T5	T8 vs T11	T3 vs T6	T9 vs T12
Sb01g001970	铵转运蛋白 AMT2	GRMZM2G338809	Os03g62200	↑	↑	↑	↑	↑	↑
Sb01g010270	谷氨酰胺合成酶 GS	GRMZM2G024104	Os03g50490	↓	--			↓	--
Sb01g029470	低亲和硝酸盐转运蛋白 NRT1/PTR	GRMZM2G161459	Os10g40600			↓	↓
Sb02g025100	谷氨酰胺合成酶 GS	GRMZM2G036783	Os09g25610	--	↑			↑	--
Sb02g033100	谷氨酰胺合成酶 GS	GRMZM2G459854				↓	↓
Sb03g035270	低亲和硝酸盐转运蛋白 NRT1/PTR	GRMZM2G035790		↓	--	↓	--	↓	--
Sb03g039530	亚硝酸还原酶 NiR	GRMZM2G367668				↓	--
Sb03g041190	低亲和硝酸盐转运蛋白 NRT1/PTR	GRMZM2G470454	Os01g65100	↑	↓			↓	--
Sb03g043020	低亲和硝酸盐转运蛋白 NRT1/PTR	GRMZM2G419328				↑	--
Sb04g002810	谷草转氨酶 AST		Os02g04170					--	↓
Sb04g031320	硝酸盐转运蛋白 NRT1/PTR		Os02g46460	↑	--
Sb05g002760	亚硝酸还原酶 NiR			--	↓
Sb06g020180	高亲和硝酸盐转运蛋白 NRT3	GRMZM2G163494	Os04g40410			↓	--
Sb06g028990	谷氨酸合成酶 GOGAT	GRMZM2G076239	Os04g53210					↓	--
Sb10g026090	低亲和硝酸盐转运蛋白 NRT1/PTR			↓	↓	↓	↓	↓	↓

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