关键词: 毛竹, Rubisco活化酶基因, 亚细胞定位, 表达模式, 原位基因编辑

Abstract:

Objective: Bamboo is an important forest plant resource with highly efficient photosynthetic carbon fixation ability. In photosynthesis, CO₂ fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygen (Rubisco), while Rubisco activase (Rca) acts as a catalytic chaperone of Rubisco and facilitates CO₂ fixation. To clarify the function of Rcas in bamboo photosynthesis, the Rca genes in Phyllostachys edulis (moso bamboo), Bambusa amplexicaulis, and Olyra latifolia were identified and analyzed systematically across the whole genomes. Furthermore, the function of PeRca1-β and PeRca2-α in moso bamboo was focused on, in order to provide reference for elucidating the molecular mechanism of Rcas in bamboo photosynthesis. Method: The known Rca amino acid sequences in rice (Oryza sativa) were used as bait, BLASTP was performed in the genome databases of P. edulis, B. amplexicaulis, and O. latifolia to obtain homologous sequences of Rca in three different bamboo species, and then systematic analyses were conducted using bioinformatics method. The expression patterns of Rca genes in different tissues and leaves under different light conditions were analyzed by RT-qPCR. The subcellular localization of Rcas from moso bamboo was observed by laser confocal microscopy. The co-expression network of PeRcas was constructed based on transcriptome data. The regulatory relationship between transcription factor and PeRcas was verified by dual luciferase reporter (DLR) assay. The function of PeRcas was investigated by in-planta gene editing in moso bamboo leaves. Result: In this study, three, three, and two members of Rcas gene family were identified in moso bamboo, B. amplexicaulis and O. latifolia, respectively. Among them, PeRca3-α in moso bamboo and OlaRca2-α in O. latifolia had longer introns, and they were clustered into an independent branch in the phylogenetic tree, which might have evolved new functions. The subcellular localization experiment in tobacco epidermal cells confirmed that PeRcas were located in chloroplasts. RT-qPCR results showed that the expression levels of PeRcas in different tissues of moso bamboo were significantly different and their response to lighting time and intensity were also diverse. Among them, PeRca1-β and PeRca2-α had higher expression levels in leaves and were induced by light, while weak light and darkness inhibited their expression. Co-expression network analysis revealed that both PeRca1-β and PeRca2-α had higher correlation with PeGLK1-1, and DLR assay confirmed that PeGLK1-1 could directly bind to the promoters and activated their expression. After in-planta gene editing, the expression levels of PeRca1-β and PeRca2-α in bamboo leaves were significantly decreased, and both the photosynthetic electron transfer rate and actual photosynthetic quantum yield were significantly reduced. Conclusion: There are three Rca members in both moso bamboo and B. amplexicaulis, and two Rca members in O. latifolia, exhibiting high conservation in evolution. The expression levels of PeRcas are significantly higher in green tissues of moso bamboo, and are induced by light and inhibited by weak light and darkness. The expression of PeRca1-β and PeRca2-α is regulated by PeGLK1-1. In-planta gene editing can reduce the expression levels of PeRca1-β and PeRca2-α in moso bamboo leaves, leading to a decrease in photosynthetic efficiency and light energy utilization. These results provide evidence for revealing the function of Rcas in bamboo and have important reference value for fully elucidating the molecular mechanism of efficient photosynthetic carbon fixation in bamboo.

Key words: Phyllostachys edulis, Rubisco activase, subcellular localization, expression pattern, in-planta gene editing