关键词: 毛竹, β-淀粉酶, PeBAM3, 转基因水稻, 淀粉分解

Abstract:

Objective: Starch produced by photosynthesis is temporarily stored in leaves during the day, and then degraded into maltose and glucose at night to provide energy for plant growth. β-amylase (BAM) is one of key enzymes in starch degradation. In this study, the function and molecular regulation mechanism of PeBAM3 were studied to clarify the function of BAM gene in the degradation of starch in moso bamboo (Phyllostachys edulis) leaves, so as to provide reference for the role of BAM gene in rapid growth of moso bamboo. Method: The diurnal variation of starch content in moso bamboo leaves was determined by anthrone colorimetry. A homologous gene PeBAM3 in the genome of moso bamboo was identified by BLAST using rice (Oryza sativa) OsBAM5 as bait. The coding region and promoter sequences of PeBAM3 were cloned and systematically analyzed by bioinformatics software. Based on transcriptome data of moso bamboo, the expression patterns of PeBAM3 in different tissues, shoots at different heights and shoots at different times within a day were analyzed. The co-expression network of PeBAM3 was constructed using the BambooNET public database, to screen the target transcription factor (TF). The real-time fluorescence quantitative PCR (qPCR) was used to detect the daily variations in expression of the TF and PeBAM3. The dual luciferase reporter assay (DLR) was applied to verify the regulatory relationship between the TF and PeBAM3. The function of PeBAM3 was validated by heterologous expression in rice. Result: The starch content in moso bamboo leaves reached its maximum value at the end of the day, and gradually decreases at night. The bamboo BAM gene PeBAM3, identified through screening, has an open reading frame of 1 635 bp, encoding 544 amino acids with a complete β-amylase conserved domain. The molecular weight of PeBAM3 is 59.50 kDa, with an isoelectric point of 6.48, an average hydrophobicity of –0.270, a protein instability index of 44.85, and a lipid index of 74.19. The promoter region of PeBAM3 (1 771 bp) contains a variety of regulatory elements related to light, stress, and hormones. Phylogenetic analysis showed that PeBAM3 is closely related to OsBAM5, suggesting that it might play an important role in starch degradation. Transcriptomic data analysis showed that PeBAM3 was relatively highly expressed in bamboo leaves and shoot buds. As the shoot height increased, the expression levels of PeBAM3 continuously rose. The expression of PeBAM3 responded to the change of photoperiod, rising at night and decreasing during the day. Co-expression network analysis revealed that the AP2/ERF (APETALA2/ethylene-responsive factor) TF gene PeERF1 was co-expressed with PeBAM3. The qPCR results demonstrated that their expression levels rose at night and decreased during the day, which was opposite to the starch content changing trend in leaves. DLR results indicated that PeERF1 was able to enhance the expression of PeBAM3. Compared with wild type, overexpression of PeBAM3 in rice led to dwarfism, reduced growth vigor in the aerial parts of transgenic rice plants, significantly increased β-amylase activity, significantly reduced starch content, and significantly increased glucose, fructose, and sucrose levels in transgenic rice leaves. Conclusion: The starch content in moso bamboo leaves shows a diurnal variation, rising in the day and falling at night. The expression level of PeBAM3 is inversely correlated with the starch content in leaves. PeERF1 can enhance the expression of PeBAM3. Overexpression of PeBAM3 promotes the degradation of starch in transgenic rice leaves. The results not only provide an important reference for in-depth analysis of the role of BAM in starch metabolism of moso bamboo, but also provide potential genetic resources for bamboo molecular breeding.

Key words: Phyllostachys edulis, β-amylase, PeBAM3, genetically modified rice, starch degradation