番茄独脚金内酯合成关键基因ccd7、ccd8rna沉默载体的构建.docx

番茄独脚金内酯合成关键基因 CCD7 CCD8 RNA沉默 载体的构建 DOI：10.14088/jki.issn0439-8114.2016.21.058 Construction of Strigolactones Key Genes CCD7、CCD8of Tomato RNA Silencing Expression Vector TIAN Fang，YAOZhao-qun，CHENMei-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 ) ： 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-PCRamplification ， 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 CCD7-8gene was inserted into pUCCRNAvi ector 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 CCD7and CCD8two concatenation genes RNA silencing expression vector p35-In-7-8. 列( Orobanche coerulescens Steph. )是一类无叶绿素并 以全寄生方式寄生于其他植物根部获取所需养分的植物， 其可寄 生在茄科、豆科、菊科、伞形科、葫芦科等多种重要经济作物上， 造成寄主植物减产5%- 100%已在亚洲、欧洲、非洲、南美洲 和北美洲的较多国家和地区发生， 每年给农业生产造成的经济损 失达数十亿美元 [1-3] 。人们采用农业、物理、化学、生物等各 项措施防治列当，均无法完全防治，在列当严重发生地区的农民 会因为防效和防治成本等问题直接放弃所种作物甚至弃耕土地 [4 ，5] 。列当靠种子进行繁殖，且种子产生量大， 1株成熟植株 可产生 5万-50万粒种子，种子可随风、流水、人为活动等因 素快速传播，可在土壤中存活数十年，这是列当难以防治的重要 原因[1 ，3] 。列当种子内的 KAI2 蛋白是一个受体蛋白，可感受 到寄主植物根系分泌的独脚金内酯类似物的刺激然后诱导列当 种子萌发 [6] ，若能减少寄主植物分泌的独脚金内酯或使其不分 泌，则有望培育出抗列当的寄主品种 [7]。Dor等[8]发现番茄 (Lycopersicon esculentum Mill.