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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (1): 40-47.doi: 10.3724/SP.J.1006.2022.01103

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• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Alternative splicing analysis of wheat glutamine synthase genes

WEI Yi-Hao1(), YU Mei-Qin2, ZHANG Xiao-Jiao1, WANG Lu-Lu1, ZHANG Zhi-Yong1, MA Xin-Ming1, LI Hui-Qing2, WANG Xiao-Chun1,2,*()   

  1. 1Collaborative Innovation Center of Henan Grain Crops, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2Department of Biochemistry, College of Life Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2020-12-29 Accepted:2021-04-14 Online:2022-01-12 Published:2021-06-28
  • Contact: WANG Xiao-Chun E-mail:yc_yihao@163.com;xiaochun.w@163.com
  • Supported by:
    National Natural Science Foundation of China(32071956);Agriculture Research System of Henan Province(S2010-01-G04)

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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

Table 1

Composition of nutrient solution with different nitrogen sources (mmol L-1)"

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

Table 2

List of primers for RT-PCR"

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
TaGS1;1-6B-3 TaGS1;1-6B-3-F GCCGGAGTTGTCATCGTGG 65 460
TaGS1;1-6B-3-R CAGACACCCTGATGACGACG
TaGS1;1-6B-4 TaGS1;1-6B-4-F CCTCAGTCAGCCGGCCAT 59 708
TaGS1;1-6B-4-R GATGATTACACGACGAGAAGGTAGC
TaTEF1 TaTEF1-F GGTGACAACATGATTGAGAGGTCC 60 678
TaTEF1-R AACAGCCACAGTTTGCCTCATG

Fig. 1

Location of TaGS genes on chromosome in wheat The position shown by the arrows are the chromosomal centromere."

Fig. 1

Location of TaGS genes on chromosome in wheat The position shown by the arrows are the chromosomal centromere."

Table 3

Differences of TaGS transcripts in IWGSC RefSeq v2.0 and the third-generation sequencing"

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
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.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

Table 3

Differences of TaGS transcripts in IWGSC RefSeq v2.0 and the third-generation sequencing"

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
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.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

Fig. 2

Comparison of gene structure of TaGS alternative splicing"

Fig. 2

Comparison of gene structure of TaGS alternative splicing"

Fig. 3

PCR amplification of TaGS1;1-6A-1, and TaGS1;1-6B-4 transcripts M: marker. The arrow points to the target segment."

Fig. 3

PCR amplification of TaGS1;1-6A-1, and TaGS1;1-6B-4 transcripts M: marker. The arrow points to the target segment."

Fig. 5

Expression of TaGS1;1 alternative splicing transcripts under different nitrogen conditions"

Fig. 5

Expression of TaGS1;1 alternative splicing transcripts under different nitrogen conditions"

Fig. 4

Functional domain of proteins corresponding to transcripts of TaGS"

Fig. 4

Functional domain of proteins corresponding to transcripts of TaGS"

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