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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (4): 966-977.doi: 10.3724/SP.J.1006.2023.21023

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

Screening of active LTR retrotransposons in wheat (Triticum aestivum L.) seedlings and analysis of their responses to abiotic stresses

ZHOU Bin-Han1(), YANG Zhu1, WANG Shu-Ping1, FANG Zheng-Wu1, HU Zan-Min2, XU Zhao-Shi3, ZHANG Ying-Xin1,*()   

  1. 1College of Agriculture, Yangtze University, Hubei Collaborative Innovation Center for Grain Industry, Jingzhou 434025, Hubei, China
    2Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
    3Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 100081, China
  • Received:2022-03-29 Accepted:2022-07-21 Online:2023-04-12 Published:2022-08-22
  • Contact: *E-mail: zhangyingxin1985@126.com
  • Supported by:
    National Key Research and Development Programs of China(2017YFD0100800);National Major Project for Developing New GM Crops(2018ZX0800909B);Key Research and Development Program of Hubei Province(2022BBA0035);Major Project for Special Technology Innovation of Hubei Province(2018ABA085)

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Abstract:

LTR (long terminal repeat) retrotransposable elements account for more than 60% of rye genome. It is of great significance to screen active LTR retrotransposons from wheat genome and analyze their transposition, the relative expression levels, and methylation levels under abiotic stress in exploring the role of retrotransposable elements in improving wheat anti-stress ability. Four active LTR retrotransposons (Fatima, Wis, Angela, and Babara) with complete structure were selected from the TREP database by bioinformatics analysis. The relative expression levels, methylation dynamics, and transposition activity of the four LTR retrotransposons were then analyzed at seedling stage (two leaves with one heart) leaves and roots of the stress-treated (NaCl, ABA, H2O2, and PEG) wheat seedlings by qRT-PCR, methylation-specific polymerase chain reaction (MSP), and transposon display method. The results suggested that the four retrotransposons had basic transcription activity under normal conditions, which could be changed under stress conditions, and their variation tendency was similar in the same stress conditions. The up-regulation of Fatima, Angela, and Babara was due to their down-regulated methylation levels, while Wis was opposite. The 3′LTR of LTR retrotransposons contained many stress-responsive cis-elements, but the regulatory effect of which were not obvious under stress treatments. Compared with that in leaves, the four LTR retrotransposons in roots were more sensitive to stresses and had higher transposition activity. This study will help to further reveal the response regularity of LTR retrotransposons under stress conditions and accumulate data for improving of stress resistance breeding wheat by using transposable elements.

Key words: wheat (Triticum aestivum L.), abiotic stress, LTR retrotransposons, the relative expression level, transposition

Table 1

Primers for qRT-PCR"

基因
Gene name		 正向引物
Forward sequence (5′-3′)		 反向引物
Reverse sequence (5′-3′)
TaActin CTCCCTCACAACAACCGC TACCAGGAACTTCCATACCAAC
Fatima AAAAAGACCAGGCTGCAACG GCGCTGATATGTTGTGCTGG
Wis CGACTTGTGGCGAAGAGTTTG GCGATGCTTAAGTCCGTCAG
Angela GTTGGCGATTGCCGCATTTT TCAACAAAACCTTCCGGTTGC
Babara GGATTACGTTGCTGCTGAGG AGCAGCTGTGCCAAATTCCT

Table 2

Primers for MSP"

引物名称
Primer name		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
M-Fatima GGTTCGAATTTGGGTAAAAATATC AACATCCTTCCTAATAACGACGAC
U-Fatima GGTTCGAATTTGGGTAAAAATATC AAACATCCTTCCTAATAACAACAAC
M-Wis TAAGATTGAATAAGGTATTGGGATATC GACTCTCTAATCAACCAACAACGA
U-Wis AATAAGATTGAATAAGGTATTGGGATATT CAACTCTCTAATCAACCAACAACAA
M-Angela GATATTTTGTTGGATCGGAGTTC AAAAATAATCGTACGTCTTCACGAT
U-Angela GATATTTTGTTGGATTGGAGTTTG AAAAATAATCATACATCTTCACAAT
M-Babara TATTTAAGTGAGGTAAGTTTTTCGT AATAATACTATCATACATCCACGTT
U-Babara TATTTAAGTGAGGTAAGTTTTTTGT AATAATACTATCATACATCCACATT

Table 3

Primers for TD"

引物名称
Primer name		 序列
Sequence (5'-3')		 引物名称
Primer name		 序列
Sequence (5'-3')
Fatima-1 CTCCCTCAGTCAGTGTCAAAA M-CA GACGATGAGTCCTGAGTAACA
Fatima-2 GGTCCCGAACTGTGCGTCT M-CC GACGATGAGTCCTGAGTAACC
Wis-1 ATGATTGTTGTTCGTCCTAT M-CG GACGATGAGTCCTGAGTAACG
Wis-2 CTCCCTCAGTCAGTGTCAAAA M-CT GACGATGAGTCCTGAGTAACT
Angela-1 ACAATTCTAGCATGAATAA M-GA GACGATGAGTCCTGAGTAAGA
Angela-2 AACTTTATTATTGCCTCTA M-GC GACGATGAGTCCTGAGTAAGC
Babara-1 TTAGCCAATCCTTTGTCCT M-GG GACGATGAGTCCTGAGTAAGG
Babara-2 TTGAAGGAGTTCCCACAAC M-GT GACGATGAGTCCTGAGTAAGT
M-0 GACGATGAGTCCTGAGTAA M-TA GACGATGAGTCCTGAGTAATA
M-AA GACGATGAGTCCTGAGTAAAA M-TC GACGATGAGTCCTGAGTAATC
M-AC GACGATGAGTCCTGAGTAAAC M-TG GACGATGAGTCCTGAGTAATG
M-AG GACGATGAGTCCTGAGTAAAG M-TT GACGATGAGTCCTGAGTAATT
M-AT GACGATGAGTCCTGAGTAAAT

Fig. 1

Structural analysis of four LTR retrotransposons in wheat GAG: group specific antigen; AP: aspartic proteases; INT: integrase; RT: the reverse transcriptase; RH: RNaseH."

Fig. 2

Relative expression pattern of LTR retrotransposon in leaves and roots of wheat seedlings under different abiotic stresses A: Fatima; B: Wis; C: Angela; D: Babara; Mock: control. Tukey method is used to test, *, **, and *** indicate significantly different at P < 0.05, P < 0.01, and P < 0.001, respectively."

Table 4

Cis-acting elements related to stress response in wheat retrotransposons"

转座子
Exist in Tes		 胁迫应答
Stress response		 顺式
作用因子
CREs		 序列(核苷酸)
Sequence
(nucleotides)		 参考文献
Reference
Fatima 光学响应元件 Light response element Box 4 ATTAAT
无氧反应响应元件 Anaerobic reaction response element ARE AAACCA
Wis 脱落酸响应元件 Cis-acting element involved in abscisic acid responsiveness ABRE ACGTG [26]
ABRE TACGTGTC [27]
干旱响应元件 Drought response element MYC CATGTG [30]
MYC CANNTG [31]
光学响应元件 Light response element ACE GACACGTATG
G-Box CACGTT
G-box CACGAC
无氧反应响应元件 Anaerobic reaction response element ARE AAACCA
Angela 脱落酸响应元件 Cis-acting element involved in abscisic acid responsiveness DRE1 ACCGAGA [28]
LTRE CCGAC [29]
干旱响应元件 Drought response element MYC CATGTG [30]
MYC CANNTG [31]
LTRE CCGAC [29]
光学响应元件 Light response element G-box CACGAC
低温响应元件 Low temperature response element LTR CCGAAA
Babara 脱落酸响应元件 Cis-acting element involved in abscisic acid responsiveness ABRE ACGTG [26]
DRE1 ACCGAGA [28]
干旱响应元件 Drought response element MYC CATGTG [30]
MYC CANNTG [31]
光学响应元件 Light response element G-Box CACGTT
无氧反应响应元件 Anaerobic reaction response element ARE AAACCA
低温响应元件 Low temperature response element LTR CCGAAA
赤霉素响应元件 Gibberellin response element TATC-box TATCCCA

Fig. 3

Comparison of retrotransposon methylation level in leaves and roots of wheat seedlings under different abiotic stresses A: Fatima; B: Wis; C: Angela; D: Babara; m: marker; M: methylation band; U: demethylation band."

Fig. 4

Transposition activity of Fatima and Wis in leaves and roots of wheat seedlings M: marker; 1, 5: the salt stress treatment. 2, 6: ABA stress treatment. 3, 7: H2O2 stress treatment. 4, 8: the drought stress treatment. The bands labelled by black arrows indicate new transposition sites in wheat genome. AA, AT, TT, CG, GG, AC, AG, CA, CT, GT, TC, TG, TA, CC, GC, and GA represent the last two bases of the amplication primers used in TD, respectively."

Fig. 5

Transposition activity analysis of Angela and Babara in leaves and roots of wheat seedlings M: marker; 1, 5: the salt stress treatment. 2, 6: ABA stress treatment. 3, 7: H2O2 stress treatment. 4, 8: the drought stress treatment. The bands labelled by black arrows indicate new transposition sites in wheat genome. AA, AT, TT, CG, GG, AC, AG, CA, CT, GT, TC, TG, TA, CC, GC, and GA represent the last two bases of the amplication primers used in TD, respectively."

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