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Acta Agronomica Sinica ›› 2026, Vol. 52 ›› Issue (1): 14-27.doi: 10.3724/SP.J.1006.2026.54037

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

Functional study of the BnGCL1 gene in ramie (Boehmeria nivea L.) in response to drought stress

Liu Hai-Bo1,2(), Zhang Lei1,2, Wang Li-Qi1,2, Shi Xiao-Li1,2, Zhou Wen-Ying1,2, Cui Guo-Xian1,2,*(), She Wei1,2,*()   

  1. 1Ramie Research Institute of Hunan Agricultural University, Changsha 410128, Hunan, China
    2College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China
  • Received:2025-03-18 Accepted:2025-10-30 Online:2026-01-12 Published:2025-11-05
  • Contact: *E-mail: weishe@hunau.edu.cn; E-mail: gx-cui@163.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(Fibre, CARS-16-E11)

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Abstract:

Drought is one of the major environmental stresses that affects plant growth and development. In this study, the functional role of the BnGCL1 gene in the drought stress response of ramie (Boehmeria nivea L.) was investigated. The results showed that the gene contains a maximum open reading frame (ORF) of 1581 bp, encoding a protein of 526 amino acids. The predicted protein has an isoelectric point of 5.79, a molecular weight of 59,123.98 Da, a fat index of 78.78, and an instability index of 37.42, indicating that it is a stable protein. BnGCL1 is expressed in the roots, stems, and leaves of ramie, and its expression is induced by drought stress. Under drought conditions, transgenic plants overexpressing BnGCL1 exhibited significantly greater root length, fresh weight, chlorophyll a, and chlorophyll b contents compared to wild-type plants. In addition, the activities of antioxidant enzymes such as APX and γ-GCL, as well as the levels of osmotic regulators including GSSG and Pro, were significantly altered. Overexpression of BnGCL1 also markedly upregulated the expression of drought-responsive genes, including AtGST1, AtGST11, AtNCED3, and AtWRKY40, suggesting that BnGCL1 enhances drought tolerance by modulating the antioxidant defense system and drought-responsive signaling pathways. Gene silencing experiments using VIGS technology further confirmed that suppression of BnGCL1 reduces drought tolerance in ramie, highlighting its critical role in drought response. This study reveals the important function of BnGCL1 under drought stress and provides a theoretical foundation for elucidating the molecular mechanisms of drought tolerance and for breeding drought-resistant ramie varieties.

Key words: Boehmeria nivea L., BnGCL1, drought stress, antioxidant enzymes, molecular mechanism

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 引物用途
Primer purpose
Actin-F1 GCTCCGTTGAACCCTAAG 苎麻内参引物
Boehmeria nivea L. reference gene
Actin-R1 GCTCCGATTGTGATGATTT
BnGCL1-F CGGCCAGTTTGAGCTTAGTG BnGCL1引物
BnGCL1 primer
BnGCL1-R TTCCTCGGCAACAGCTTTGA
VIGS-BnGCL1-F gtgagtaaggttaccgaattcATGGCACTCATTTCGCAGACA BnGCL1-VIGS引物
BnGCL1-VIGS primer
VIGS-BnGCL1-R cgtgagctcggtaccggatccTTAGTAGAGTAGCTCCTCAAAAACAGG
VIGS-BnPDS-F gtgagtaaggttaccgaattcCTCAAAAACTCAAACGACTCTTCG BnPDS-VIGS引物
BnPDS-VIGS primer
VIGS-BnPDS-R cgtgagctcggtaccggatccGTTTGTTAGCGGCTTCCTGC
AtACTIN8-F TCAGCACTTTCCAGCAGATG 拟南芥内参基因
AtACTIN8-R CTGTGGACAATGCCTGGAC Arabidopsis thaliana reference gene
AtGST1-F ACAAGAATTGCTCTGGCGGA AtGST1引物
AtGST1 primer
AtGST1-R CGGAGGGAAGGAGAGGGTTA
AtGST11-F ATGGCAGGAATCAAAGTTTT AtGST11引物
AtGST11 primer
AtGST11-R TTAAGAACCTTCTTAGCAGA
AtNCED3-F CTCAGCCGCCATTATCGTCT AtNCED3引物
AtNCED3 primer
AtNCED3-R GAGGAGTGTGAAGCGCAGAT
AtWRKY40-F TGGCTTAAACCGCCACATCT AtWRKY40引物
AtWRKY40 primer
AtWRKY40-R ATTCTTGACGTTGGGCTCGT
BnActin-F TCGAAGAGAGGTATCCTTAC 苎麻内参基因
BnActin-R CTCGTTGTAGAAAGTGTGAT Boehmeria nivea L. reference gene
BnGCL1-F AATTGGTTTCCAGCCTAAAT BnGCL1基因
BnGCL1-R TTTTGGCATGTAGTTTCTCA BnGCL1 primer

Fig. 1

Cloning of BnGCL1 gene M: DNA marker; 1, 2: cloning product of BnGCL1."

Fig. 2

Homology comparison of amino acid sequences between the BnGCL1 and GCL proteins from other plants Black indicates 100% sequence homology, pink indicates homology greater than 75%, and blue indicates homology greater than 50%."

Fig. 3

Phylogenetic tree of BnGCL1 and GCL proteins from other plants"

Fig. 4

Expression levels of BnGCL1 gene in ramie leaves under drought stress CK: untreated (no 20% PEG-200 simulated drought stress); 20% PEG: treated with 20% PEG-200 to simulate drought stress. Different lowercase letters on the error bars indicate significant differences at the 5% level."

Fig. 5

Growth of Arabidopsis thaliana after overexpressing BnGCL1 under drought stress Treatments are the same as those given in Fig. 4. WT: wild-type Arabidopsis strain; BnGCL1-OE5, BnGCL1-OE10: overexpressed Arabidopsis lines."

Fig. 6

Root length and fresh weight of Arabidopsis thaliana after overexpressing BnGCL1 under drought stress Treatments are the same as those given in Fig. 4. Abbreviations are the same as those given in Fig. 5. Different lowercase letters on the error bars indicate significant differences at the 5% level."

Fig. 7

Contents of chlorophyll a and chlorophyll b in BnGCL1-overexpressing Arabidopsis thaliana under drought stress Treatments are the same as those given in Fig. 4. Abbreviations are the same as those given in Fig. 5. Different lowercase letters on the error bars indicate significant differences at the 5% level."

Fig. 8

Antioxidant enzyme activities in BnGCL1-overexpressing Arabidopsis thaliana under drought stress A: superoxide dismutase (SOD) activity; B: content of malondialdehyde (MDA); C: content of hydrogen peroxide (H2O2); D: content of oxidized glutathione (GSSG); E: activity of ascorbic acid peroxidase (APX); F: γ-glutamyl cysteine synthase (γ-GCL) activity. Different lowercase letters on the error bars indicate significant differences at the 5% level. Treatments are the same as those given in Fig. 4. Abbreviations are the same as those given in Fig. 5."

Fig. 9

Proline (Pro) and glutathione (GSH) contents in BnGCL1-overexpressing Arabidopsis thaliana under drought stress Treatments are the same as those given in Fig. 4. Abbreviations are the same as those given in Fig. 5. Different lowercase letters on the error bars indicate significant differences at the 5% level."

Fig. 10

Expression levels of AtGST1, AtGST11, AtNCED3, and AtWRKY40 genes in BnGCL1-overexpressing Arabidopsis thaliana under drought stress Treatments are the same as those given in Fig. 4. Abbreviations are the same as those given in Fig. 5. Different lowercase letters on the error bars indicate significant differences at the 5% level."

Fig. 11

Verification of the drought resistance function of BnGCL1 using VIGS experiments A: whitening phenotype of pTRV::BnPDS lines. B: MDA content of VIGS silenced lines and control after normal treatment and drought treatment. C: relative expression levels of BnGCL1 gene between VIGS silent lines and control lines. D: the control group and VIGS silent lines under normal treatment. E: leaf phenotypes between control group and VIGS silent lines after seven days of drought treatment. F: leaf phenotypes between control group and VIGS silent lines after ten days of drought treatment. Scale = 2 cm. Different lowercase letters on the error bars indicate significant differences at the 5% level."

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