茶树CsPAL3基因的亚细胞定位及遗传转化

苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)由多基因家族编码,是花青素等多酚物质合成途径的起始酶,对其合成具有调控作用。以紫化茶树武夷奇种C18为材料,采用Gateway技术体系分别构建了茶树的CsPAL3过表达载体pGWB502:CsPAL3和pGWB505:CsPAL3:GFP,并成功将其转入根癌农杆菌GV3101。注射烟草瞬时表达激光共聚焦扫描显微镜可观察到GFP绿色荧光,结果表明CsPAL3主要集中在细胞核和细胞膜中。通过侵染拟南芥,筛选纯合子,获得稳定表达的转CsPAL3基因拟南芥。实时荧光定量PCR(qPCR)检测发现,CsPAL3在转CsPAL3基因拟南芥中的根部表达量显著高于叶片,且CsPAL3基因受光照调控。该结果为进一步研究茶树CsPAL3基因功能以及促进茶树花青素合成与积累的分子调控机理提供科学依据。

Subcellular Localization and Genetic Transformation of CsPAL3 Gene of Tea Plants(Camellia sinensis)

Phenylalanine ammonia lyase (PAL) is encoded by a multigene family and is the starting enzyme for the synthesis of polyphenolic substances such as anthocyanins, and PAL has a regulatory effect on anthocyanins synthesis. The Gateway technology system was used to construct the tea plant CsPAL3 overexpression vector pGWB502: CsPAL3 and pGWB505: CsPAL3: GFP from purple leaf tea cultivar Wuyi qizhong C18 (Camellia sinensis), and successfully transferred into Agrobacterium tumefaciens GV3101. By transient expression of the injected tobacco, GFP green fluorescence was observed by laser confocal scanning microscopy, indicating successful transient expression, and the results showed that the CsPAL3 is mainly concentrated in the nucleus and cell membrane. Through invading Arabidopsis thaliana, the homozygotes were screened to obtain stably expressed transgenic CsPAL3 A. thaliana. The expression of CsPAL3 in the transgenic CsPAL3 gene A. thaliana was significantly higher than that in the leaves by real time quantitative PCR (qPCR), and the CsPAL3 gene was regulated by light. This study provides a scientific basis for further study of the function of CsPAL3 gene in tea tree and the molecular regulation mechanism of tea anthocyanin synthesis and accumulation.