月季漆酶基因RcLAC15克隆与表达分析

Cloning and expression analysis of the laccase gene RcLAC15 from Rosa chinensis

摘要:

漆酶(laccase,LAC)属于铜蓝蛋白氧化酶家族，与植物木质素合成及响应生物与非生物胁迫等生物学功能密切相关，但目前关于月季漆酶基因的研究尚未见报道。皮刺对月季的管理和采摘造成极大的不便，已成为月季育种关注的重点。为了探究漆酶基因在月季中的表达模式，本研究以古老月季品种‘月月粉’(Rosa chinensis ‘Old Blush’)为材料，克隆了1个漆酶基因并将其命名为RcLAC15。基因表达分析结果表明，RcLAC15在月季皮刺中的表达量显著高于在根、茎、叶等器官中的表达量。在月季基因组中鉴定了58个漆酶基因，生物信息学分析发现RcLAC15是AtLAC15的同源基因，预测RcLAC15为稳定的亲水蛋白，不具备跨膜结构。将重组表达载体pBI121-proRcLAC15::GUS转化拟南芥，GUS染色结果表明RcLAC15启动子在拟南芥叶片边缘特异性驱动了GUS基因表达。本研究发现RcLAC15在月季皮刺特异性表达，为探索漆酶基因在月季皮刺中的生物学功能提供了参考。

关键词:月季;RcLAC15;皮刺特异性表达;GUS染色

Abstract:

Laccases (LACs), belonging to the multicopper oxidase family, are closely associated with various biological functions including lignin synthesis and responses to biotic and abiotic stresses in plants. However, few studies have reported the laccase genes in China rose (Rosa chinensis). Prickles cause difficulties to the management and harvest of R. chinensis and have become a trait concerned in the breeding. To investigate the expression patterns of laccase genes in roses, we cloned a laccase gene from an ancient variety R. chinensis ‘Old Blush’ and named it RcLAC15. The expression level of RcLAC15 in prickles was significantly higher than those in roots, stems, and leaves. Fifty-eight laccase genes were identified in the genome of R. chinensis, and bioinformatics analysis revealed that RcLAC15 was a homolog of AtLAC15, predicting that RcLAC15 was a stable hydrophilic protein without transmembrane structures. The recombinant expression vector pBI121-proRcLAC15::GUS was introduced into Arabidopsis, and GUS staining results showed that the RcLAC15 promoter specifically drove GUS gene expression at the edges of Arabidopsis leaves. In summary, RcLAC15 is a gene specifically expressed in the prickles of R. chinensis. This discovery provides a reference for exploring the biological functions of laccase genes in the prickles of R. chinensis.

Key words:Rosa chinensis;RcLAC15;prickle-specific expression;GUS staining