番茄红素β环化酶基因(Lyc-β)RNAi载体构建及表达鉴定  

Construction of RNA Interference Vector for Lycopene Cyclase β Gene(Lyc-β) and its Expression Identification in Lycopersicun esculentum

作　　者：马超[1] 马兵钢[1] 郝青南[1] 何娟[1] 鲁晓燕[1] 吴志苹[1] 

机构地区：[1]石河子大学农学院园艺系,新疆生产建设兵团绿洲生态农业重点实验室,石河子832003

出　　处：《农业生物技术学报》2010年第1期10-17,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(30460081);新疆兵团博士资金项目(ZD2007JC06)共同资助

摘　　要：从番茄(Lycopersicun esculentumMill)中提取总RNA,根据GenBank中番茄红素β环化酶基因(Lyc-β)序列(X86452),经mRNA反转录扩增出2段Lyc-β基因高度保守的300bpDNA片段,从β-葡萄糖苷酶基因(gusA)中扩增出170bp的内含子片段。分别将两段不同的Lyc-β片段的正、反向序列与内含子连接,构建出两套干扰Lyc-β基因的植物表达载体。经根癌农杆菌(Agrobacterium tumefaciems)介导转化番茄,22株转基因植株通过荧光定量PCR分析干扰效果,结果显示,2套干扰载体的干扰效率表现出一定的差异,Lyc-β基因mRNA平均含量分别为对照的11.78%和13.86%,进一步用HPLC分析转化株的番茄红素含量,结果表明,转基因植株中番茄红素的含量最高可达13.84μg/gFW,是对照的4.26倍。The total RNA was isolated from tomato（Lycopersicun esculentum Mill）.Two fragments of 300 bp of lycopene cyclase β gene（Lyc-β） were reversely transcribed and amplified from its mRNA according to GenBank sequence（X86452）.One intron fragment of 170 bp was amplified from β-glucosidase gene（gusA）.Two plant expression vectors for interference Lyc-β gene were constructed by ligating the intron fragment of gusA gene with two Lyc-β fragments which were designed in different direction,respectively.The plasmids were transformed into tomato through leaf disc method mediated by Agrobacterium tumefaciems.The twenty-two transgenic tomato plants were obtained,then real time PCR assays were performed to analyse the interference effect among the two RNAi vectors.The results demonstrated that the different interference efficacy was reflected on the mRNA abundance of Lyc-β gene,which was in average of 11.78% and 13.86%,respectively compared with the control of wild plant.The lycopene contents were analyzed by HPLC from transgenic tomato plants,and the highest content was 13.84 μg/g FW,4.26 fold compared with control.

关 键 词：番茄 类胡萝卜素 番茄红素 番茄红素β环化酶基因 RNAI 

分 类 号：S571.1[农业科学—茶叶生产加工]