Cloning and expression analysis of PeLAC in Phyllostachys edulis
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摘要: 漆酶（LAC）对植物生长发育具有重要作用。本研究以毛竹（Phyllostachys edulis（Carr.） Lehaie）为实验材料，克隆获得毛竹漆酶基因PeLAC的cDNA和基因组序列，长度分别为1692bp和2785bp。该基因包含5个内含子和6个外显子；PeLAC蛋白编码563个氨基酸，推测的分子质量和等电点分别为62.3kD和9.056。系统进化分析表明，PeLAC与其它植物的漆酶具有较高的一致性。经预测PeLAC基因编码序列中含有miR397的靶点，利用RLM-5'RACE技术证明miR397对PeLAC能够准确切割，位点位于靶序列的第10~11位碱基之间。组织特异性分析表明，PeLAC基因在茎中表达丰度最高，根中次之，叶鞘中较少，叶片中几乎未检测到表达；随着笋高度的增加，PeLAC表达丰度上升，生长至15cm时达到最大值，30cm时又有所下降；而miR397的表达与PeLAC相反。本研究同时克隆了PeLAC基因上游启动子序列PeLACp，其包含ABRE、MBS等多种与逆境胁迫相关的响应元件。利用ABA（100μmol/L）和NaCl（400mmol/L）溶液处理可明显诱导PeLAC在毛竹根中表达，而GA3（100μmol/L）处理则对其表达具有抑制作用。Abstract: 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.
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Keywords:
- Phyllostachys edulis /
- LAC /
- miR397 /
- Expression analysis
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