Cloning and expression analysis of PeLAC in Phyllostachys edulis

Laccase (LAC) has many functions in plants, playing important roles in both growth and development. The cDNA, genomic DNA, and promoter sequences of PeLAC were isolated from Moso bamboo (Phyllostachys edulis(Carr.) Lehaie) using PCR techniques. The open reading frame (ORF) and genomic DNA sequences of PeLAC in Ph. edulis were 1692 bp and 2785 bp, respectively. There were six exons separated by five introns in the PeLAC genomic sequence, which were in full compliance with the intron splicing principle of GT-AG. PeLAC encoded 563 amino acid residues with a calculated molecular weight of 62.3 kD and theoretical isoelectric point of 9.056. Phylogenetic analysis demonstrated that PeLAC had high identities with LACs from other plants. Prediction showed that a putative miR397-targeted site was found in the coding region of PeLAC, which was further validated by RLM-5' RACE. The sequencing result of cleavage products showed that the cleavage site was at the 10th base of miR397 complementary to PeLAC in Ph. edulis, confirming that miR397 can guide mRNA cleavage of PeLAC. Tissue specific expression analysis showed that PeLAC was expressed at the highest level in the stem, followed by the root and leaf sheath, but hardly at all in the leaf blade. With increasing shoot height, the expression abundance of PeLAC increased to a maximum at 15 cm, and then declined at 30 cm; whereas the expression of miR397 showed the opposite pattern. The promoter sequence of PeLAC was also isolated, and was 2000 bp and included many responsive elements such as ABRE and MBS related to abiotic stresses, indicating that PeLAC might be involved in responses to hormones and drought. Further experiments confirmed that PeLAC was obviously upregulated in roots under ABA (100 μmol/L for 24 h) and NaCl (400 mmol/L for 4 h) treatment, but was downregulated after treatment with GA₃ (100 μmol/L for 3 h). The isolation and expression analysis of PeLAC presented in this research has laid a foundation for further study on the biological functions of LACs in Ph. edulis.