Main Content of the Study on the Function of the Ramie BnGCL1 Gene in Response to Drought Stress

I. Research Background and Objectives

• Drought is a major abiotic stress that affects plant growth; it inhibits growth, reduces photosynthetic efficiency, and causes metabolic disorders. As a unique bast fiber crop in China, ramie (Boehmeria nivea L.) is highly drought-tolerant, but drought still affects its yield and quality.
• Glutamate-cysteine ligase (GCL), also known as γ-glutamyl cysteine synthetase (γ-GCS), catalyzes the biosynthesis of glutathione (GSH). GSH plays a key role in plant antioxidant defense and maintaining redox homeostasis, although research on plant GCL genes remains limited.
• This study aims to investigate the functional mechanism of the ramie BnGCL1 gene under drought stress, thereby providing a theoretical basis and a candidate gene for research on the molecular mechanisms underlying ramie drought resistance and for the breeding of drought-resistant varieties.

II. Research Materials and Methods

(I) Experimental Materials

• Ramie variety: Zhongzhu No. 1; Arabidopsis thaliana Col-0 and related vectors (pCAMBIA1300-35S-BnGCL1-myc, etc.) were stored in the laboratory.
• Agrobacterium tumefaciens GV3101 and Escherichia coli strains TOP10 and DH5α were purchased from Shanghai Weidi Biotechnology Co., Ltd.

(II) Core Research Methods

1. Bioinformatics analysis: Tools such as NCBI and ExPASy were used to analyze the ORF, the physicochemical properties of the protein, conserved domains, and species homology of the BnGCL1 gene.
2. Gene expression analysis: 20% PEG-200 was used to simulate drought stress, and qPCR was used to detect the expression level of the BnGCL1 gene in ramie leaves at different time points.
3. Overexpression verification: An overexpression vector was constructed and transformed into Arabidopsis thaliana. After screening positive lines, the phenotype, physiological indicators, and expression of drought-resistance-related genes in plants under drought stress were determined.
4. Silencing verification: The pTRV-VIGS system was used to silence the BnGCL1 gene in ramie, and changes in plant phenotype and MDA content under drought stress were observed.

III. Key Research Findings

(I) Characteristics of the BnGCL1 Gene

• Gene sequence: The ORF is 1,581 bp long and encodes 526 amino acids; the protein has an isoelectric point of 5.79, a molecular weight of 59,123.98 Da, is a stable protein, and contains a conserved PLN02611 domain.
• Homology: The amino acid sequence similarity with Nicotiana tabacum, Arabidopsis thaliana, and other plants exceeds 80%, and it is most closely related to Arabidopsis thaliana and Brassica juncea.
• Expression pattern: It is expressed in the roots, stems, and leaves of ramie, and the expression level peaks at 12 hours of drought stress, reaching 3.62 times the level observed before treatment.

(II) Drought Resistance Effect of Overexpressing BnGCL1

• Phenotype: Under drought stress, the root length of the overexpressed Arabidopsis thaliana increased by 28.92%–57.23%, and the fresh weight increased by 81.42%–131.19%, indicating better growth than the wild type.
• Photosynthetic pigments: The chlorophyll a content increased by 25.07%–99.09%, and the chlorophyll b content increased by 11.14%–47.26%, resulting in more stable photosynthetic capacity.
• Physiological indicators: The activities of antioxidant enzymes such as APX and γ-GCL were significantly increased; GSSG levels rose; and SOD activity improved, enhancing the ability to scavenge reactive oxygen species. Meanwhile, Pro and GSH levels decreased, which is thought to be related to metabolic redistribution or consumption.
• Gene expression: It significantly upregulated drought-responsive genes such as AtGST1, AtGST11, AtNCED3, and AtWRKY40, which are involved in antioxidant and ABA signaling pathways.

(III) Effects of Silencing BnGCL1

• Under drought stress, the leaves of the silenced lines showed more pronounced wilting, and the MDA content was significantly higher than that of the control, indicating a significant decrease in drought resistance, which confirms the drought-resistant function of BnGCL1.

IV. Research Conclusions

The BnGCL1 gene contributes to the plant’s drought stress response by enhancing antioxidant enzyme activity, regulating the metabolism of osmotic adjustment substances, and upregulating the expression of drought-responsive genes, thereby significantly improving the plant’s drought resistance. This gene serves as an important candidate gene for the molecular breeding of drought-resistant ramie and also provides a reference for improving the drought resistance of other crops.