摘要
通过BLAST分析,获得了番茄漆酶(Laccase,LAC)基因相关的15个EST片段,并对这些EST进行了同源比较和序列拼接,得到1个含有小分子RNA mi R397识别位点的LAC片段,命名为Le LACmi R397。根据蛋白质结构域推测,Le LACmi R397蛋白存在1个Cu–氧化酶结构域。Le LACmi R397时空表达分析表明,其在番茄根、花、成熟果实和愈伤组织中特异性表达,而在叶中不表达。根据该片段的核苷酸序列信息进行5′-和3′-RACE扩增,得到了番茄LAC基因的全长cD NA(LeL ACmiR 397,登录号EU503151),其在番茄基因组中对应的基因为Solyc07g049460.2.1。通过在番茄中过表达mi R397a基因,发现转基因番茄中Le LACmi R397表达量降低,同时其PPO、POD、SOD含量也有所下降。用丁香酸和芥子酸处理转基因植株幼苗,其根系比非转基因植株更长,抗性增强,表明Le LACmi R397与番茄植株的抗性反应有关。
Laccases are glycoproteins with polyphenol oxidase acitivity depending on copper. They ubiquitously exist in plants,fungi,insects,bacteria and other organisms,playing crucial roles in lignin synthesis,ion absorption and stress responses. It has been documented that several LAC genes were regulated by micro RNAs at post–transcriptional level. In this study,homology BLAST was conducted in Gen Bank for LAC ESTs. Resultly,15 tomato ESTs highly similar with LAC genes were obtained,and spliced into a LAC fragment named as Le LAC^miR397 containing a recognition site of mi R397. In addition,a Cu-oxidase domain was predicted to be in the deduced Le LAC^miR397 protein. To check the temporal and spatial expression of Le LAC^miR397,semi-quantitative RT-PCR was performed. It was found that Le LAC^miR397 specifically expressed in roots,flowers,ripe fruit and callus,but not in leaves. Then,its full length c DNA was cloned with RT-PCR and 5′- and 3′-RACEs,and registered as EU503151 in Gen Bank,corresponding to Solyc07g049460.2.1 in tomato genome. The data showed that Le LAC^miR397 expression was down-regulated in the transgenic tomato overexpressing mi R397 a,and the contents of PPO,POD and SOD were reduced too. When being treated with Syringic acid and Sinapic acid,transgenic tomato seedlings overexpressing Le LAC^miR397 had longer root system and showed stronger resistance than WT. These results suggested that Le LAC^miR397 was related to resistance to Syringic acid and Sinapic acid in tomato.
出处
《园艺学报》
CAS
CSCD
北大核心
2015年第7期1285-1298,共14页
Acta Horticulturae Sinica
基金
国家自然科学基金项目(31171946)
教育部长江学者和创新团队发展计划项目(IRT1155)
关键词
番茄
漆酶
LAC
克隆
抗性
tomato
laccase
LAC
cloning
Le LACmi R397gene
resistance
