番茄独脚金内酯合成关键基因CCD7CCD8 RNA沉默载体构建.docVIP

番茄独脚金内酯合成关键基因CCD7CCD8 RNA沉默载体构建 　　摘要：根据已知的番茄（Lycopersicon esculentum Mill.）CCD7和CCD8基因的序列设计特异性引物，采用RT-PCR克隆CCD7和CCD8基因片段，通过特定酶切将2个基因进行拼接，再将串联基因以反向重复的方式连入pUCCRNAi载体，并定向插入到p35植物表达载体上。结果表明，克隆获得了大小约为400 bp的CCD7和CCD8基因片段，基因片段拼接后获得800 bp左右的串联片段CCD7-8；将该基因片段插入pUCCRNAi载体后得到1 700 bp大小的含Intron的反向重复序列In-7-8，并成功插入到植物表达载体p35中，通过酶切分析，RNAi载体构建正确。最终成功构建了番茄CCD7和CCD8 2个串联基因的RNA沉默表达载体p35-In-7-8。 　　关键词：番茄（Lycopersicon esculentum Mill.）；独脚金内酯；列当；类胡萝卜素裂解双加氧酶7（CCD7）和8（CCD8）；RNA沉默载体 　　中图分类号：Q78 文献标识码：A 文章编号：0439-8114（2016）21-5668-04 　　DOI：10.14088/j.cnki.issn0439-8114.2016.21.058 　　Construction of Strigolactones Key Genes CCD7、CCD8 of 　　Tomato RNA Silencing Expression Vector 　　TIAN Fang， YAO Zhao-qun， CHEN Mei-xiu， XU Ying， ZHAO Si-feng 　　（Key Laboratory of Universities of Xinjiang Uygur Autonomous Region for Oasis Agricul-tural Pest Management and Plant Protection Resource Utilization /College of Agriculture， Shihezi Uni-versity， Shihezi 832003， Xinjiang， China） 　　Abstract： In order to construct tomato（Lycopersicon esculentum Mill.） carotenoid cleavage dioxygense 7（CCD7） and 8 （CCD8） RNA silencing expression vector， the specific primers were designed according to the published sequence of tomato CCD7 and CCD8. The CCD7 and CCD8 fragments were obtained by RT-PCR amplification，then they were concatenated through specific enzyme digestion，and cloned into pUCCRNAi vector in a inverted repeat manner，finally inserted into p35 plant expression vector. Results show that we cloned about 400 bp CCD7 and CCD8 gene fragment，respectively. The concatenation gene CCD7-8 of 800 bp was constructed by gene fragment splicing. Two copies of CCD7-8 gene was inserted into pUCCRNAi vector and got 1 700 bp contain Intron’s inverted repeat sequence In-7-8. Then they were inserted into p35 vector successfully and the RNAi vector was constructed correctly by enzyme digestion analysis. Finally successful constructed tomato CCD7 and CCD8 two concatenation genes’ RNA silencing expression vector p35-In-7-8.