Dissection of Key Regulatory Pathways for Low-temperature Tolerance and Functional Verification of TaGGCT18-6A Gene in Wheat

doi:10.7668/hbnxb.20195902

Abstract

Abstract:

To elucidate the mechanisms of cold response and explore superior cold-tolerance gene resources in wheat,this study employed transcriptome sequencing to uncover key regulatory networks underlying wheat cold response,and performed functional validation of the candidate gene TaGGCT18-6A.The results demonstrated that 4 ℃ cold treatments induced 10 893 and 18 784 differentially expressed genes(DEGs)in wheat seedlings after 6 h and 24 h cold treatment,respectively.KEGG analysis revealed significant enrichment of DEGs in pathways including MAPK signaling transduction and glutathione metabolism.A γ-glutamyl cyclotransferase gene TaGGCT18-6A was cloned through screening key genes in glutathione metabolism.This gene had a length of 1 772 bp,encoding 218 amino acids with conserved GGCT-like superfamily and ChaC core domain.Promoter cis-acting element analysis identified stress-responsive elements such as low-temperature-responsive(LTR)and dehydration-responsive (DRE)elements; consistently,expression pattern analysis showed sustained upregulation of TaGGCT18-6A under 4 ℃ cold treatments. Functional validation in transgenic rice revealed that overexpression lines OE#1, OE#2 and OE#3 exhibited significantly enhanced survival rate,plant height,and biomass. Overexpression lines OE#1 and OE#2 exhibited significantly enhanced plant height, while overexpression lines OE#1, OE#2 and OE#3 exhibited significantly reduced relative electrolyte leakage under -4 ℃ freezing treatments.After 4 ℃ treatment,overexpression lines accumulated higher levels of osmolytes(proline and soluble sugars),decreased malondialdehyde(MDA)content,and increased activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT).These findings collectively demonstrated that TaGGCT18-6A enhanced plant cold tolerance by regulating glutathione metabolism to improve antioxidant capacity.This study will provide a theoretical foundation and valuable genetic resources for molecular breeding of cold-tolerant wheat.

Key words: Wheat, Low temperature stress, Transcriptome analysis, γ-glutamyl cyclotransferase, TaGGCT18-6A

CLC Number:

CHENG Chunhua, CHEN Tao, ZHANG Long, GUO Lijian, CHE Zhuo, MA Jingfu, YANG Delong. Dissection of Key Regulatory Pathways for Low-temperature Tolerance and Functional Verification of TaGGCT18-6A Gene in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 1-11. doi: 10.7668/hbnxb.20195902.

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References 47

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引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
TaGGCT18-6A-F	TCAGCAGGATGGTGCTGTG	TaGGCT18-6A基因编码区PCR
TaGGCT18-6A-R	TCAATGGGAGAATGTCAAGG
TaGGCT18-6A-Os-F	ACTATCCTTCGCAAGACCCTT	TaGGCT18-6A转基因水稻PCR
TaGGCT18-6A-Os-R	GGAGTGGATACGAAAACTAATACAT
TaGGCT18-6A-qRT-PCR-F	CGTTCACCGGTGGTCIATTT	TaGGCT18-6A荧光定量PCR
TaGGCT18-6A-qRT-PCR-R	GGCTCCACTGTTCCTTGAGA
TaMAPKKK6-1A -qRT-PCR-F	GATGGTGCACGGGGACGTC	TaMAPKKK6-1A荧光定量PCR
TaMAPKKK6-1A-qRT-PCR-R	ACAACCATGCAGCCGAGCG
TaMAPKK7-4A-qRT-PCR-F	CTTCGCGCTCAAGCTCTTC	TaMAPKK7-4A荧光定量PCR
TaMAPKK7-4A-qRT-PCR-R	ACACGAGCTCCAGCGCCAG
TaMAPK5-4A-qRT-PCR-F	CAACGCCTTCGACAACAACAT	TaMAPK5-4A荧光定量PCR
TaMAPK5-4A-qRT-PCR-R	GTCCGTGTCCATGAGCTCAG
TaGGT-2D-qRT-PCR-F	CTGTGACCTCGCCGCACG	TaGGT-2D荧光定量PCR
TaGGT-2D-qRT-PCR-R	GTGAGCCTGACGAGCATGAA
TaGST6-5A-qRT-PCR-F	CTCGGCGGCAAGGATTTCT	TaGST6-5A荧光定量PCR
TaGST6-5A-qRT-PCR-R	CCTGGGTTTCAGCCTCTCC
TaActin-F	GTGTCGCACCAGAGGATCAT	小麦内参基因荧光定量PCR
TaActin-R	CGCTGGCATACAAGGACAGA

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
TaGGCT18-6A-F	TCAGCAGGATGGTGCTGTG	TaGGCT18-6A基因编码区PCR
TaGGCT18-6A-R	TCAATGGGAGAATGTCAAGG
TaGGCT18-6A-Os-F	ACTATCCTTCGCAAGACCCTT	TaGGCT18-6A转基因水稻PCR
TaGGCT18-6A-Os-R	GGAGTGGATACGAAAACTAATACAT
TaGGCT18-6A-qRT-PCR-F	CGTTCACCGGTGGTCIATTT	TaGGCT18-6A荧光定量PCR
TaGGCT18-6A-qRT-PCR-R	GGCTCCACTGTTCCTTGAGA
TaMAPKKK6-1A -qRT-PCR-F	GATGGTGCACGGGGACGTC	TaMAPKKK6-1A荧光定量PCR
TaMAPKKK6-1A-qRT-PCR-R	ACAACCATGCAGCCGAGCG
TaMAPKK7-4A-qRT-PCR-F	CTTCGCGCTCAAGCTCTTC	TaMAPKK7-4A荧光定量PCR
TaMAPKK7-4A-qRT-PCR-R	ACACGAGCTCCAGCGCCAG
TaMAPK5-4A-qRT-PCR-F	CAACGCCTTCGACAACAACAT	TaMAPK5-4A荧光定量PCR
TaMAPK5-4A-qRT-PCR-R	GTCCGTGTCCATGAGCTCAG
TaGGT-2D-qRT-PCR-F	CTGTGACCTCGCCGCACG	TaGGT-2D荧光定量PCR
TaGGT-2D-qRT-PCR-R	GTGAGCCTGACGAGCATGAA
TaGST6-5A-qRT-PCR-F	CTCGGCGGCAAGGATTTCT	TaGST6-5A荧光定量PCR
TaGST6-5A-qRT-PCR-R	CCTGGGTTTCAGCCTCTCC
TaActin-F	GTGTCGCACCAGAGGATCAT	小麦内参基因荧光定量PCR
TaActin-R	CGCTGGCATACAAGGACAGA

样品 Sample	原始序列数 Raw reads	过滤后序列数 Clean reads	过滤后碱基数/Gb Clean bases	Q30/%	GC含量/% GC content	比对率/% Total mapped ratio
CK1	55 064 908	54 452 354	8.17	94.12	55.78	90.83
CK2	54 300 614	53 691 934	8.05	94.15	56.15	90.99
CK3	55 233 878	54 503 334	8.18	93.91	58.20	89.95
Cold-6 h-1	57 758 566	56 960 080	8.54	94.29	56.38	91.17
Cold-6 h-2	55 003 544	53 988 202	8.10	94.33	56.27	91.64
Cold-6 h-3	55 752 342	54 775 938	8.22	94.20	55.29	89.52
Cold-24 h-1	55 233 518	54 487 646	8.17	93.73	57.33	89.28
Cold-24 h-2	54 561 382	53 976 740	8.10	94.05	56.44	90.17
Cold-24 h-3	53 870 550	53 342 274	8.00	93.77	55.47	90.68

样品 Sample	原始序列数 Raw reads	过滤后序列数 Clean reads	过滤后碱基数/Gb Clean bases	Q30/%	GC含量/% GC content	比对率/% Total mapped ratio
CK1	55 064 908	54 452 354	8.17	94.12	55.78	90.83
CK2	54 300 614	53 691 934	8.05	94.15	56.15	90.99
CK3	55 233 878	54 503 334	8.18	93.91	58.20	89.95
Cold-6 h-1	57 758 566	56 960 080	8.54	94.29	56.38	91.17
Cold-6 h-2	55 003 544	53 988 202	8.10	94.33	56.27	91.64
Cold-6 h-3	55 752 342	54 775 938	8.22	94.20	55.29	89.52
Cold-24 h-1	55 233 518	54 487 646	8.17	93.73	57.33	89.28
Cold-24 h-2	54 561 382	53 976 740	8.10	94.05	56.44	90.17
Cold-24 h-3	53 870 550	53 342 274	8.00	93.77	55.47	90.68

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