Abstract：Glutamate cysteine ligase (GCS) is a key rate-limiting enzyme in the biosynthesis of glutathione (GSH), a crucial thiol compound that plays a vital role in plant response to abiotic stresses such as cadmium (Cd). To investigate the biological function of the GCS gene in rapeseed (Brassica napus) under Cd stress, the Cd hyperaccumulator variety 'Nanyou 868' was used in this study. The complete coding sequence (CDS) of the GCS gene was cloned by RT-PCR and named as BnaGCS (GenBank No. PV694275). Sequence analysis revealed that the ORF of BnaGCS was 1 536 bp in length, encoding 511 amino acids. The pBWA(V)HS-BnaGCS-1209-osgfp overexpression vector was constructed and introduced into Nicotiana benthamiana via Agrobacterium tumefaciens-mediated transformation. Molecularly confirmed transgenic plants were subjected to Cd stress, and gene expression was analyzed, and both cadmium content and GSH content were measured. qRT-PCR results showed that under Cd stress, the transcription level of BnaGCS in both roots and shoots of transgenic tobacco was significantly up-regulated (P<0.05), with a higher up-regulation amplitude in roots than in shoots. Cd and GSH content measurements revealed that Cd stress significantly increased Cd content in both roots and shoots of wild-type (WT) and transgenic plants (P<0.05). The Cd content in roots of transgenic tobacco was significantly lower than that in WT, while the Cd content in shoots was significantly higher than that in WT (P<0.05). Under Cd stress, the GSH content in both wild-type and transgenic tobacco significantly increased, while the GSH content in transgenic tobacco was significantly lower than that in WT (P<0.05). These findings suggested that BnaGCS might be involved in the response of transgenic tobacco to Cd stress. This study provides a theoretical foundation for elucidating the function of BnaGCS and the mechanism of glutathione biosynthesis.
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