小麦谷氨酰胺合成酶基因可变剪接分析

doi:10.3724/SP.J.1006.2022.01103

Abstract

Abstract:

As a key enzyme for nitrogen assimilation in plant, glutamine synthetase (TaGS) is encoded by 12 genes in common wheat (Triticum aestivum L.), namely TaGS1;1-6A/6B/6D, TaGS1;2-4A/4B/4D, TaGS1;3-4A/4B/4D, and TaGS2-2A/2B/2D. Transcripts of TaGS genes were obtained using the single molecular sequencing technology, and the results showed that TaGS1;1-6A had one alternative splicing (AS) version of transcripts and TaGS1;1-6B had two AS versions. Compared with the TaGS encoded by normal transcript, TaGS1;1-6A-1 lacked part of the Gln_synt_N domain, and TaGS1;1-6B-3 lacked the Gln_synth_gly_rich_ site domain; TaGS1;1-6B-4 was a new AS event, and encoded a truncated GS without Gln_synth_gly_rich_site and Gln_synth_ cat_dom domain. The relative expression level of TaGS alternative splicing transcripts of wheat cultivars with different nitrogen use efficiencies showed that the relative expression level of TaGS1;1-6A-1 in leaves of YM49 (N-efficient) was significantly higher than that of XN509 (N-inefficient), and increased with the increase of nitrogen supply. In roots, the relative expression of TaGS1;1-6A-1 increased with the increase of nitrogen supply in YM49, but decreased in XN509. TaGS1;1-6B-3 showed a similar expression trend in YM49 and XN509, and high concentration of NO₃^- promoted the expression of TaGS1;1-6B-3 in leaves while inhibited in roots. High concentration of NH₄⁺ inhibited the expression of TaGS1;1-6B-3 in leaves, but had little effect in roots. TaGS1;1-6B-4 was mainly expressed under low nitrogen condition, and its expression in roots of YM49 was not affected by NH₄⁺ supply. Understanding the AS transcript versions of TaGS genes is helpful to illustrate the functions of TaGS isozymes in nitrogen metabolism of wheat.

Key words: glutamine synthetase, transcription, alternative splicing, wheat, glutamine synthetase, transcription, alternative splicing

WEI Yi-Hao, YU Mei-Qin, ZHANG Xiao-Jiao, WANG Lu-Lu, ZHANG Zhi-Yong, MA Xin-Ming, LI Hui-Qing, WANG Xiao-Chun. Alternative splicing analysis of wheat glutamine synthase genes[J].Acta Agronomica Sinica, 2022, 48(1): 40-47.

Figures/Tables 14

Table 1

Table 2

Fig. 1

Table 3

Fig. 2

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References 22

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氮含量 Nitrogen content	KH₂PO₄	MgSO₄	KCl	CaCl₂	Ca(NO₃^-)₂	NH₄Cl
N0	0.2	1	1.5	2.5	0	0
0.2 mmol L^-1 NO₃^-	0.2	1	1.5	2.4	0.1	0
2 mmol L^-1 NO₃^-	0.2	1	1.5	1.5	1	0
5 mmol L^-1 NO₃^-	0.2	1	1.5	0	2.5	0
10 mmol L^-1 NO₃^-	0.2	1	1.5	0	5	0
20 mmol L^-1 NO₃^-	0.2	1	1.5	0	10	0
0.2 mmol L^-1 NH₄⁺	0.2	1	1.5	2.5	0	0.2
2 mmol L^-1 NH₄⁺	0.2	1	1.5	2.5	0	2
5 mmol L^-1 NH₄⁺	0.2	1	1.5	2.5	0	5
10 mmol L^-1 NH₄⁺	0.2	1	1.5	2.5	0	10
20 mmol L^-1 NH₄⁺	0.2	1	1.5	2.5	0	20

转录本名称 Transcript name	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	退火温度 Annealing temperature (℃)	PCR产物长度 PCR fragment (bp)
TaGS1;1-6A-1	TaGS1;1-6A-1-F	GGCCAATACCCATATTTTCACTGTG	60	1199
	TaGS1;1-6A-1-R	CAGTGGCCACCCACCAAATC	60	1199
TaGS1;1-6B-3	TaGS1;1-6B-3-F	GCCGGAGTTGTCATCGTGG	65	460
	TaGS1;1-6B-3-R	CAGACACCCTGATGACGACG	65	460
TaGS1;1-6B-4	TaGS1;1-6B-4-F	CCTCAGTCAGCCGGCCAT	59	708
	TaGS1;1-6B-4-R	GATGATTACACGACGAGAAGGTAGC	59	708
TaTEF1	TaTEF1-F	GGTGACAACATGATTGAGAGGTCC	60	678
	TaTEF1-R	AACAGCCACAGTTTGCCTCATG	60	678

TaGS同工酶 TaGS isoforms		基因编号 Gene ID	IWGSC RefSeq2.0			第3代测序分析 Third-generation sequencing analysis
TaGS同工酶 TaGS isoforms		基因编号 Gene ID	转录本编号 Transcript ID	蛋白质 Protein (aa)	分子量 MW (kD)	转录本编号 Transcript ID		蛋白质 Protein (aa)	分子量 MW (kD)	CPM^*
TaGS1;1	TraesCS6A02G298100	TraesCS6A02G298100.1	349	38.4		TraesCS6A02G298100.1	359	39.7	6.4
		TraesCS6A02G298100.2	356	39.2		TraesCS6A02G298100.2	356	39.2	103.5
	TraesCS6B02G327500	TraesCS6B02G327500.1	356	39.2		TraesCS6B02G327500.1	356	39.2	154.9
		TraesCS6B02G327500.2	325	35.8					0
		TraesCS6B02G327500.3	322	35.6		TraesCS6B02G327500.3	322	35.6	7.6
						ONT.45646.3	223	24.5	3.8
	TraesCS6D02G383600LC	TraesCS6D02G383600LC.1	356	39.2		TraesCS6D02G383600LC.1	356	39.2	16
	TraesCS6D02G383600LC	TraesCS6D02G383600LC.2	325	35.8					0
TaGS1;2	TraesCS4A02G063800	TraesCS4A02G063800.1	354	38.7		TraesCS4A02G063800.1	354	38.7	114.6
	TraesCS4B02G240900	TraesCS4B02G240900.1	354	38.7		TraesCS4B02G240900.1	354	38.7	134.2
	TraesCS4D02G240700	TraesCS4D02G240700.1	354	38.7		TraesCS4D02G240700.1	354	38.7	170
TaGS1;3	TraesCS4A02G266900	TraesCS4A02G266900.1	362	39.6		TraesCS4A02G266900.1	362	39.6	8.8
	TraesCS4B02G047400	TraesCS4B02G047400.1	362	39.5		TraesCS4B02G047400.1	362	39.5	3.3
	TraesCS4D02G047400	TraesCS4D02G047400.1	362	39.5		TraesCS4D02G047400.1	362	39.5	13
TaGS2	TraesCS2A02G500400	TraesCS2A02G500400.1	427	46.7		TraesCS2A02G500400.1	427	46.7	9.7
		TraesCS2A02G500400.2	423	46.09					0
	TraesCS2B02G528300	TraesCS2B02G528300.1	423	46.08					0
		TraesCS2B02G528300.2	369	40.63		TraesCS2B02G528300.2	427	46.7	53
	TraesCS2D02G500600	TraesCS2D02G500600.1	427	46.7		TraesCS2D02G500600.1	427	46.7	27.3

TaGS同工酶 TaGS isoforms		基因编号 Gene ID	IWGSC RefSeq2.0			第3代测序分析 Third-generation sequencing analysis
TaGS同工酶 TaGS isoforms		基因编号 Gene ID	转录本编号 Transcript ID	蛋白质 Protein (aa)	分子量 MW (kD)	转录本编号 Transcript ID		蛋白质 Protein (aa)	分子量 MW (kD)	CPM^*
TaGS1;1	TraesCS6A02G298100	TraesCS6A02G298100.1	349	38.4		TraesCS6A02G298100.1	359	39.7	6.4
		TraesCS6A02G298100.2	356	39.2		TraesCS6A02G298100.2	356	39.2	103.5
	TraesCS6B02G327500	TraesCS6B02G327500.1	356	39.2		TraesCS6B02G327500.1	356	39.2	154.9
		TraesCS6B02G327500.2	325	35.8					0
		TraesCS6B02G327500.3	322	35.6		TraesCS6B02G327500.3	322	35.6	7.6
						ONT.45646.3	223	24.5	3.8
	TraesCS6D02G383600LC	TraesCS6D02G383600LC.1	356	39.2		TraesCS6D02G383600LC.1	356	39.2	16
	TraesCS6D02G383600LC	TraesCS6D02G383600LC.2	325	35.8					0
TaGS1;2	TraesCS4A02G063800	TraesCS4A02G063800.1	354	38.7		TraesCS4A02G063800.1	354	38.7	114.6
	TraesCS4B02G240900	TraesCS4B02G240900.1	354	38.7		TraesCS4B02G240900.1	354	38.7	134.2
	TraesCS4D02G240700	TraesCS4D02G240700.1	354	38.7		TraesCS4D02G240700.1	354	38.7	170
TaGS1;3	TraesCS4A02G266900	TraesCS4A02G266900.1	362	39.6		TraesCS4A02G266900.1	362	39.6	8.8
	TraesCS4B02G047400	TraesCS4B02G047400.1	362	39.5		TraesCS4B02G047400.1	362	39.5	3.3
	TraesCS4D02G047400	TraesCS4D02G047400.1	362	39.5		TraesCS4D02G047400.1	362	39.5	13
TaGS2	TraesCS2A02G500400	TraesCS2A02G500400.1	427	46.7		TraesCS2A02G500400.1	427	46.7	9.7
		TraesCS2A02G500400.2	423	46.09					0
	TraesCS2B02G528300	TraesCS2B02G528300.1	423	46.08					0
		TraesCS2B02G528300.2	369	40.63		TraesCS2B02G528300.2	427	46.7	53
	TraesCS2D02G500600	TraesCS2D02G500600.1	427	46.7		TraesCS2D02G500600.1	427	46.7	27.3

Alternative splicing analysis of wheat glutamine synthase genes

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