茶树漆酶基因CsLAC4和CsLAC12的克隆与表达分析
Cloning and expression analysis of the laccase genes CsLAC4 and CsLAC12 from the tea plant

为探究漆酶（laccase，LAC）基因在茶树防御植食性昆虫为害中的作用，以龙井43茶树为材料，采用反转录PCR（reverse transcription-polymerase chain reaction，RT-PCR）技术克隆得到了2条漆酶基因CsLAC4和CsLAC12的完整cDNA序列，并通过实时荧光定量RCR技术测定其在茶树不同组织中的表达模式以及不同处理下表达量的变化。结果表明，茶树漆酶基因CsLAC4和CsLAC12的序列全长分别为1 665 bp和1 743 bp，分别编码555个和581个氨基酸残基。预测蛋白分子量分别为61.1 kD和63.9 kD，理论等电点分别为8.34和9.18；均具有植物漆酶的3个典型离子结合域；在系统进化树上聚在不同分支。CsLAC4和CsLAC12均在茶树茎中相对表达量最高，分别为2.62和1.33，CsLAC4在茶树花和种子中相对表达量最低，分别为0.58和0.66，CsLAC12在茶树种子中相对表达量最低，仅为0.20；与对照相比，CsLAC4的相对表达量能够在茉莉酸处理3 h、机械损伤和茶尺蠖Ectropis obliqua幼虫取食处理12、24 h时被显著诱导；但CsLAC12的相对表达量在茉莉酸处理12 h和机械损伤处理12、24 h时被显著抑制，茶尺蠖幼虫取食处理6 h时能够诱导其相对表达量升高。表明CsLAC4和CsLAC12在茶树防御植食性昆虫为害中作用不同，但都在对茶尺蠖取食的响应中具有重要作用。
To investigate the role of laccase (LAC) genes in the defense of tea plant against herbivorous insect pests, two complete cDNA sequences of LACs were cloned by reverse transcription polymerase chain reaction (RT-PCR) from Longjing 43, named as CsLAC4 and CsLAC12. The expression patterns of two LACs in different tissues and their changes after induced by different treatments were analyzed by real-time quantitative PCR (qPCR). The results showed that the full-length cDNA sequences of CsLAC4 and CsLAC12 were 1 665 bp and 1 743 bp, encoding 555 and 581 amino acid residues, respectively. The predicted molecular weights of CsLAC4 and CsLAC12 proteins were 61.1 kD and 63.9 kD, with a theoretical isoelectric point of 8.34 and 9.18, respectively. The protein sequences of CsLAC4 and CsLAC12 all contained three typical copper binding domains, and CsLAC4 and CsLAC12 were clustered in different clades in phylogenetic tree. The qPCR analyses showed that CsLAC4 and CsLAC12 both had the highest expression level in the stems of tea plant (2.62 and 1.33, respectively). CsLAC4 had the lowest expression level in flowers and seeds of tea plant (0.58 and 0.66, respectively). CsLAC12 had the lowest expression level in seeds of tea plant (0.20). Compared with the control, the expression level of CsLAC4 was increased significantly after exogenous jasmonic acid treatment for three hours, mechanical damage and Ectropis obliqua larval infestation for 12 h and 24 h. The expression level of CsLAC12 was significantly suppressed after exogenous jasmonic acid treatment for 12 h and mechanical damage for 12 h and 24 h, but E. obliqua infestation for six hours significantly induced higher expression level of CsLAC12. The results indicated that CsLAC4 and CsLAC12 probably played different roles in tea plants against herbivorous insect pests, but they both played an important role in the induced defense response to the infestation of E. obliqua