Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (4): 617-634.doi: 10.3864/j.issn.0578-1752.2025.04.001

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

Cloning and Heat Tolerance Function of Wheat TaGRAS34-5A Gene

DIAO DengChao1(), LI YunLi1, MENG XiangYu1, JI SongHan1, SUN YuChen1, MA XueHong1, LI Jie1, FENG YongJia1, LI ChunLian1, WU JianHui1,3, ZENG QingDong2,3, HAN DeJun1,3, $\boxed{\hbox{WANG ChangFa}}$1,3, ZHENG WeiJun1,3()   

  1. 1 College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi
    2 College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi
    3 Northwest A&F University/National Key Laboratory for Crop Stress Resistance and High- Efficiency Production, Yangling 712100, Shaanxi
  • Received:2024-07-26 Accepted:2024-10-11 Online:2025-02-16 Published:2025-02-24
  • Contact: ZHENG WeiJun

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Abstract:

【Objective】The GRAS family constitutes a unique class of plant-specific transcription factors that play a pivotal role in plant development and stress response. To elucidate the function of GRAS family genes in wheat heat tolerance，which can provide genetic resources and theoretical foundation for wheat heat-resistant breeding.【Method】A potential heat stress-responsive transcription factor gene, TaGRAS34-5A, was identified through transcriptome analysis of TAM107 and Chinese spring wheat seedlings under high-temperature conditions. Subsequently, a bioinformatics analysis was performed on TaGRAS34-5A, and a phylogenetic tree was constructed to elucidate its molecular characteristics. The expression pattern of TaGRAS34-5A under various stresses, including high temperature, abscisic acid (ABA), ethylene (ETH), and salicylic acid (SA) treatments, were examined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. The subcellular localization of the TaGRAS34-5A protein was determined using wheat protoplast transient expression technique. Furthermore, the heat tolerance function of TaGRAS34-5A was validated using the heterologous expression system of Saccharomyces cerevisiae and the BSMV:VIGS (Barley stripe mosaic virus: Virus-Induced Gene Silencing) silencing technique. potential interacting proteins of TaGRAS34-5A were screened using yeast two-hybrid technology, and the heat tolerance function was verified, providing preliminary insights into its heat tolerance mechanism.【Result】TaGRAS34-5A, equipped with a characteristic GRAS domain and belongs to the GRAS transcription factor family, is localized to both the cell nucleus and cytoplasm. Bioinformatics analysis indicates that the TaGRAS34-5A promoter contains a large number of hormone response elements and light response elements, and it is most closely related to TaSCL14, OsGRAS23, and AtSCL14 in terms of phylogenetic relationships, suggesting its potential function in responding to oxidative stress. Its expression is upregulated under high-temperature, ethylene (ETH), abscisic acid (ABA), and salicylic acid (SA) treatments, peaking at 4, 6, 0.5, and 12 hours post-treatment, respectively, with the most significant induction observed under heat stress and SA. Functional assays in yeast demonstrated that heterologous expression of TaGRAS34-5A enhances the heat tolerance of the yeast. The results of BSMV:VIGS transient silencing experiment showed that after the 42 ℃ high-temperature treatment, TaGRAS34-5A silenced plants exhibited decreased chlorophyll content, reduced POD enzyme activity, increased cellular peroxidation, and decreased heat tolerance compared to the control. Preliminary studies on the heat tolerance mechanism suggest that TaGRAS34-5A exhibits strong transcriptional self-activation activity.it may modulate wheat heat tolerance by interacting with proteins such as the bZIP family transcription factor HBP-1b and the E3 ubiquitin ligase hel2, thereby regulating cellular redox homeostasis and detoxification processes, positively influencing the heat tolerance of wheat.【Conclusion】TaGRAS34-5A is induced by heat, ABA, ETH, and SA, and its encoded protein is located in the nucleus and cytoplasm. It exhibits transcriptional activation activity. Heterologous overexpression of TaGRAS34-5A enhances the heat tolerance of Saccharomyces cerevisiae. Silencing TaGRAS34-5A in wheat plants increases cellular peroxidation, decreases chlorophyll content, and reduces heat tolerance. TaGRAS34-5A may regulate the heat tolerance of wheat by modulating cellular redox state and detoxification processes.

Key words: wheat, GRAS transcription factor, heat stress, gene silencing, protein interaction

Table 1

Primers used in this experiment"

引物名称Primer name 引物序列Primer sequence (5′-3′)
TaGRAS34-5A(1-88)F TCAGAGGAGGACCTGCATATGATGGGCTCCTCCTCCTAC
TaGRAS34-5A(1-88)R TCGACGGATCCCCGGGAATTCACGAGGTCCTCCGGCGT
TaGRAS34-5A(88)F TCAGAGGAGGACCTGCATATGCTGCCCTTCATCTCCCGC
TaGRAS34-5A(88)R TCGACGGATCCCCGGGAATTTTATGAGCTAGCATCGTT
TaGRAS34-5A(1-199)F TCAGAGGAGGACCTGCATATGATGGGCTCCTCCTCCTAC
TaGRAS34-5A(1-199)R TCGACGGATCCCCGGGAATTGAGGAAGGCGGAGCTCAT
TaGRAS34-5A(199)F TCAGAGGAGGACCTGCATATGAAGGGGATGCAGGAGGCC
TaGRAS34-5A(199)R TCGACGGATCCCCGGGAATTTTATGAGCTAGCATCGTT
TaGRAS34-5A(1-350)F TCAGAGGAGGACCTGCATATGATGGGCTCCTCCTCCTAC
TaGRAS34-5A(1-350)R TCGACGGATCCCCGGGAATTGGCCCCCTTCCCACCGC
TaGRAS34-5A(350)F TCAGAGGAGGACCTGCATATGAATGGCAAGGGGAGGGGC
TaGRAS34-5A(350)R TCGACGGATCCCCGGGAATTTTATGAGCTAGCATCGTT
TaGRAS34-5A(FL)F TCAGAGGAGGACCTGCATATGATGGCAAGGGGAGGGGCC
TaGRAS34-5A(FL)R TCGACGGATCCCCGGGAATTTTATGAGCTAGCATCGTT
TaGRAS34-5A pTF486F CCATCAGCCCAGAGGATCCATGGGCTCCTCCTCC
TaGRAS34-5A pTF486R CCTTGCTCACCATGGATCCTGAGCTAGCATCGTT
TaGRAS34-5A BSMVF1 TTTTTTTTAGCTAGCTGATTAATTAACATCCCCGGCTCGCC
TaGRAS34-5A BSMVR1 TCTTCCGTTGCTAGCTGAGCGGCCGCTTGTCGTCGATGTCC
TaGRAS34-5A BSMVF2 TTTTTTTTAGCTAGCTGATTAATTAAACTCGTTGTTGGATT
TaGRAS34-5A BSMVR2 TCTTCCGTTGCTAGCTGAGCGGCCGCACGTCGTCTCCCAGC
TaGRAS34-5AqPCRF1 AATATGGGTTCCAGTGGCCG
TaGRAS34-5AqPCRR1 AGTGATCCTTACATCTGGCGG
TaGRAS34-5DqPCRF1 GAGGGCCGCCAGATGTAAG
TaGRAS34-5DqPCRR1 CAAATCCTCTTTGCGGAGCG
TaHBP-1b(c38)-3BqPCRF TCCATGAGTGGAAATGGGGC
TaHBP-1b(c38)-3BqPCRR ACTACGCAGCTCAGTATCGC
TaHBP-1b(c38)-3DqPCRF AGCTGCAGTTAATGCCCGTG
TaHBP-1b(c38)-3DqPCRR AGCTCAGAAGGTCGGAAACC
TraesCS5B02G347900-qPCRF TGGTTCCGATGAGGTTGAGC
TraesCS5B02G347900-qPCRR GTGTAGTGCTGCCTGGTCAT
TraesCS7D02G442700-qPCRF CACCCTCCACCTGGTTCTC
TraesCS7D02G442700qPCRR GGTCCGGTGGGATACCCT
TraesCS7D02G443100qPCRF CCCTCCACCTGGTTCTCAG
TraesCS7D02G443100qPCRR CGGTGGGATACCCTCCTT

Fig. 1

Amino acid sequence alignment diagram of wheat TaGRAS34-5A protein with five other plants"

Fig. 2

Phylogenetic tree of GRAS proteins from different species"

Fig. 3

Protein sequence alignment of TaGRAS34-5A and TaGRAS34-5D"

Fig. 4

Bioinformatics analysis of TaGRAS34-5A protein A: Protein conserved domain prediction; B: TaGRAS34-5A motif analysis; C: Protein secondary structure prediction; D: Protein tertiary structure prediction"

Fig. 5

Cis acting elements in TaGRAS34-5A promoter"

Fig. 6

Expression patterns of wheat TaGRAS34-5A under different treatments *: P<0.05, **: P<0.01, ***: P<0.001; ****: P<0.0001. The same as below"

Fig. 7

Subcellular localization of TaGRAS34-5A protein in wheat"

Fig. 8

Yeast heat tolerance test of wheat TaGRAS34-5A"

Fig. 9

Validation of heat resistance function of wheat TaGRAS34-5A based on VIGS system A: Silence efficiency detection; B: Phenotype after treatment at 42 ℃ for 16 h/38 ℃ for 8 h for 5 days; C: Total chlorophyll content; D: Peroxidase activity; E: Malondialdehyde content; F: Leaf pathogenesis map of BSMV-VIGS; G: Hydrogen peroxide, superoxide anion staining; NS: No virus inoculation and normal temperature growth. HS: No virus inoculation and high temperature treatment; BSMV: 00: Vaccination with empty virus and high-temperature treatment; BSMV: TaGRAS34-5AT1/T2: Inoculate recombinant virus and treat with high temperature. Different lowercase letters indicate significant differences at the 0.05 probability level"

Fig. 10

Transcription activation activity analysis of TaGRAS34-5A (A) and yeast dual heterozygous screening library (B)"

Table 2

List of potential interacting proteins for TaGRAS34-5A"

名称Name 位置Position (bp) 描述Description
TraesCS1D02G376000 Chr.1D:452502146-452506435(-) 丝氨酸/苏氨酸蛋白激酶STN7，叶绿体
Serine/threonine-protein kinase STN7, chloroplastic
TraesCS2B02G570100 Chr.2B:760890079-760892799(-) 泛素结合酶E2 28 Ubiquitin-conjugating enzyme E2 28
TraesCS3B02G220400 Chr.3B:269247185-269257672(+) 转录因子HBP-1b（c38） Transcription factor HBP-1b(c38)
TraesCS3B02G391000 Chr.3B:616257855-616262245(+) 加速细胞死亡蛋白6 Protein ACCELERATED CELL DEATH 6
TraesCS3D02G194800 Chr.3D:187831578-187841857(+) 转录因子HBP-1b（c38）Transcription factor HBP-1b(c38)
TraesCS3D02G352000 Chr.3D:462860336-462864221(-) 含锚蛋白重复序列蛋白At5g02620 Ankyrin repeat-containing protein At5g02620
TraesCS4D02G066600 Chr.4D:41216419-41220849(-) 含CBS结构域的蛋白质CBSX3 CBS domain-containing protein CBSX3
TraesCS5A02G193700 Chr.5A:397143531-397146861(+) 线粒体外膜孔蛋白Mitochondrial outer membrane porin
TraesCS5B02G189000 Chr.5B:341051034-341054531(+) 线粒体外膜孔蛋白Mitochondrial outer membrane porin
TraesCS5B02G347900 Chr.5B:528780525-528784513(+) E3泛素蛋白连接酶Hel2 E3 ubiquitin-protein ligase hel2
TraesCS6A02G041900 Chr.6A:22168247-22170677(+) 多酚氧化酶F，叶绿体Polyphenol oxidase F, chloroplastic
TraesCS6B02G286700 Chr.6B:516482885-516488319(-) 蛋白质TRANSPARENT TESTA GLABRA 1 Protein TRANSPARENT TESTA GLABRA 1
TraesCS7A02G514800 Chr.7A:700689132-700707173(+) 转录起始因子TFIID亚基1 Transcription initiation factor TFIID subunit 1
TraesCS7D02G442700 Chr.7D:562148061-562150169(-) 泛素Polyubiquitin
TraesCS7D02G443100 Chr.7D:562401777-562425958(+) 泛素Polyubiquitin

Fig. 11

Expression heatmap of potential interacting proteins for TaGRAS34-5A A: Transcriptome of China Spring under 7 days heat stress during seedling stage; B: Transcriptome of TAM107 under 7 days heat stress during seedling stage"

Fig. 12

Point-to-point verification of TaGRAS34-5A interacting proteins"

Fig. 13

Expression patterns of five interacting proteins of TaGRAS34-5A under heat stress"

Fig. 14

Phylogenetic tree and sequence alignment diagram of TaHBP-1b, Polyubiquitin, and its homologous proteins"

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