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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (1): 126-137.doi: 10.3724/SP.J.1006.2024.34045

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

Function analysis of an AP2/ERF transcription factor GhTINY2 in cotton negatively regulating salt tolerance

XIAO Sheng-Hua1,2,*(), LU Yan1(), LI An-Zi1, QIN Yao-Bin1, LIAO Ming-Jing1, BI Zhao-Fu1, ZHUO Gan-Feng1, ZHU Yong-Hong2, ZHU Long-Fu2,*()   

  1. 1State Key Laboratory of Conservation and Utilization of Agro-Biological Resources in Subtropical Region / College of Agriculture, Guangxi University, Nanning 530000, Guangxi, China
    2State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430000, Hubei, China
  • Received:2023-03-06 Accepted:2023-06-29 Online:2024-01-12 Published:2023-07-21
  • Contact: *E-mail: shxiao@gxu.edu.cn; E-mail: lfzhu@mail.hzau.edu.cn
  • About author:**Contributed equally to this study
  • Supported by:
    Initial Scientific Research Fund of High-level Personnel in Guangxi University(A3310051044);Guangxi University Agricultural College Research Development Fund(EE101711)

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Abstract:

Cotton is a relatively salt-tolerant crop, but high salt stress leads to a significant decline in cotton yield and fiber quality. Mining the genes involved in salt-tolerance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Here, we identified an AP2/ERF transcription factor GhTINY2 in the transcriptome database from cotton treated with salt, and the relative expression level of GhTINY2 was reduced by salt. Subsequently, the salt-resistant phenotype and physiological indicators of the GhTINY2-overexpression Arabidopsis were analyzed. The results revealed that the GhTINY2-overexpression Arabidopsis had a significant decrease in seed germination rate, the content of proline, soluble sugar, and chlorophyll under salt stress, leading to more severe leaf wilting compared with WT. RNA-seq data from GhTINY2-transgenic Arabidopsis revealed that differentially expressed genes (DEGs) were enriched in a series of biological processes, including chlorophyll metabolism and response to stimulus, and the relative expression level of these DEGs significantly was down-regulated. Moreover, the silence of GhTINY2 in cotton through Virus-induced gene silencing (VIGS) assay showed that TRV:GhTINY2 had a significant increase in chlorophyll and proline content, leading to improved salt tolerance compare with TRV:00. In conclusion, these findings suggest that GhTINY2 was an important gene in cotton that negatively regulated salt stress resistance, and it was expected to create salt-tolerant cotton materials using GhTINY2 gene by modern genetic engineering technology in the future.

Key words: cotton, GhTINY2, salt stress, transcription factor, transgenic

Table 1

Primers used for vector construction and RT-qPCR"

引物名称Primer name 引物序列Primer sequence (5°-3°)
GhUB7-F GAAGGCATTCCACCTGACCAAC
GhUB7-R CTTGACCTTCTTCTTCTTGTGCTTG
AtACTIN2-F AAATCACAGCACTTGCACCAAGC
AtACTIN2-R GGCCTTGGAGATCCACATCTGC
AtP5CS2-F ATGATCTTATTTATGTTCTGC
AtP5CS2-R CACTATCTTCCGTCACTAT
AtP5CS1-F ACCAGAAGCACGGTCATTC
AtP5CS1-R CCATCTGAGAATCTTGTG
AtProDH2-F AAGTGTCAGCATCACAAC
AtProDH2-R CACGAAGAAATCATCAC
AtRD20-F GATGGAATCGTCTATCCTTGGG
AtRD20-R ACTGGGACATACCTTCCTTCGG
AtRD22-F CCCATTCCCAACTCTCTCCAT
AtRD22-R GACCTTTTCCGCTGCCAAC
AtRD26-F ATGGGTCGTCATCGTCTTCTTC
AtRD26-R GAAACGCATCGTAACCACCG
TRV:GhTINY2-F GCGTGAGCTCGGTACCGGAGGAGCTGAGCCAGATAGTG
TRV:GhTINY2-R GCCTCCATGGGGATCCCTAGAAATTTTGACCTATCCATGCTA
GhTINY2-F CATCTTCATCGTTGTCGTCCTCA
GhTINY2-R CAGTTTTCTTCGTAATACCAAGGCAT

Fig. 1

Relative expression pattern of GhTINY2 induced by NaCl and ABA A: heat map of GhTINY2 induced by NaCl, red color represents high expression and white color represents low expression; B-C: the expression of GhTINY2 induced by NaCl (B) and ABA (C) through RT-qPCR assay. **, P < 0.01, Student’s t-test."

Fig. 2

Overexpression of GhTINY2 inhibited seed germination in Arabidopsis under salt stress A: the germination phenotypes of GhTINY2-overexpression Arabidopsis in 1/2 MS medium with or without NaCl. B-C: the germination rate of GhTINY2-overexpression seeds in 1/2 MS medium without NaCl (B) or with 0.12 mol L-1 NaCl (C). Bar: 1 cm, **, P < 0.01, Student’s t-test."

Fig. 3

Overexpression of GhTINY2 decreased resistance to NaCl in Arabidopsis A: the growth phenotypes of GhTINY2-overexpression Arabidopsis in nutrient soil with or without NaCl. B: the survival rate of GhTINY2- overexpression Arabidopsis under salt stress. C: the water loss rate of detached leaf of GhTINY2-overexpression Arabidopsis. **: P < 0.01, Student’s t-test."

Fig. 4

Determination of physiological indexes in GhTINY2-overexpression plants under salt stress A-D: determination of content of MDA (A), soluble sugar (B), proline (C), and chlorphyll (D) in GhTINY2-overexpression Arabidopsis under salt stress. **: P < 0.01, Student’s t-test."

Fig. 5

Analysis of RNA-seq in GhTINY2-overexpression Arabidopsis A: the volcano plot of differentially expressed genes (DEGs) in RNA-seq data from GhTINY2-overexpression Arabidopsis. B-C: KEGG (B) and GO (C) analysis of DEGs in RNA-seq data from GhTINY2-overexpression Arabidopsis."

Fig. 6

Heat map of differentially expressed genes in GhTINY2- overexpression Arabidopsis The numbers in the heat map represent the down regulation fold of differentially expressed genes in GhTINY2-OE compared to WT."

Fig. 7

Relative expression analysis of gene involved in salt stress response in GhTINY2-overexpression Arabidopsis A-B: the relative expression of salt stress marker genes (A) and proline synthesis/degradation genes (B) in WT and GhTINY2-overexpression Arabidopsis under normal condition and salt stress. *: P < 0.05, **: P < 0.01, Student’s t-test."

Fig. 8

Down-regulation of GhTINY2 increased salt tolerance in cotton A: the silence effect of GhTINY2 in cotton seedlings by RT-qPCR. TRV:00 represent the control cotton seedlings and TRV:GhTINY2 represent the cotton seedlings in which the expression of GhTINY2 is down-regulated. B: the phenotype of TRV:00 and TRV:GhTINY2 plant response to salt. C-D: the content of chlorophyll (C) and proline (D) in TRV:00 and TRV:GhTINY2 plant under salt stress. **: P < 0.01, Student’s t-test."

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[3] YANG Jian-Chang;ZHANG Jian-Hua;WANG Zhi-Qin;ZH0U Qing-Sen. Changes in Contents of Polyamines in the Flag Leaf and Their Relationship with Drought-resistance of Rice Cultivars under Water Deficiency Stress[J]. Acta Agron Sin, 2004, 30(11): 1069 -1075 .
[4] Yan Mei;Yang Guangsheng;Fu Tingdong;Yan Hongyan. Studies on the Ecotypical Male Sterile-fertile Line of Brassica napus L.Ⅲ. Sensitivity to Temperature of 8-8112AB and Its Inheritance[J]. Acta Agron Sin, 2003, 29(03): 330 -335 .
[5] Wang Yongsheng;Wang Jing;Duan Jingya;Wang Jinfa;Liu Liangshi. Isolation and Genetic Research of a Dwarf Tiilering Mutant Rice[J]. Acta Agron Sin, 2002, 28(02): 235 -239 .
[6] WANG Li-Yan;ZHAO Ke-Fu. Some Physiological Response of Zea mays under Salt-stress[J]. Acta Agron Sin, 2005, 31(02): 264 -268 .
[7] TIAN Meng-Liang;HUNAG Yu-Bi;TAN Gong-Xie;LIU Yong-Jian;RONG Ting-Zhao. Sequence Polymorphism of waxy Genes in Landraces of Waxy Maize from Southwest China[J]. Acta Agron Sin, 2008, 34(05): 729 -736 .
[8] HU Xi-Yuan;LI Jian-Ping;SONG Xi-Fang. Efficiency of Spatial Statistical Analysis in Superior Genotype Selection of Plant Breeding[J]. Acta Agron Sin, 2008, 34(03): 412 -417 .
[9] WANG Yan;QIU Li-Ming;XIE Wen-Juan;HUANG Wei;YE Feng;ZHANG Fu-Chun;MA Ji. Cold Tolerance of Transgenic Tobacco Carrying Gene Encoding Insect Antifreeze Protein[J]. Acta Agron Sin, 2008, 34(03): 397 -402 .
[10] ZHENG Xi;WU Jian-Guo;LOU Xiang-Yang;XU Hai-Ming;SHI Chun-Hai. Mapping and Analysis of QTLs on Maternal and Endosperm Genomes for Histidine and Arginine in Rice (Oryza sativa L.) across Environments[J]. Acta Agron Sin, 2008, 34(03): 369 -375 .
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