卷丹转基因体系构建及岷江百合LrCCoAOMT的导入  

Construction of Transformation System and Integration of LrCCoAOMT Gene into Lilium lancifolium

作　　者：符勇耀 刘建玲 朱艺勇 徐文姬 雷美艳[2] 杨利平[1] FU Yongyao;Liu Jianling;ZHU Yiyong;XU Wenji;Lei Meiyan;YANG Liping(School of Advanced Agriculture and Bioengineering,Yangtze Normal University,Chongqing 408100,China;Chongqing Institute of Medicinal Plant Cultivation,Chongqing 408435,China)

机构地区：[1]长江师范学院现代农业与生物工程学院,重庆408100 [2]重庆市药物种植研究所,重庆408435

出　　处：《园艺学报》2020年第7期1345-1358,共14页Acta Horticulturae Sinica

基　　金：国家自然科学基金青年基金项目(31500245);重庆市科技局基础研究与前沿探索项目(cstc2019jcyj-msxm X0014);重庆市教委科学技术项目(KJQN201801428);重庆市科研机构绩效激励引导专项(cstc2018jxjl-jbky130015);重庆市人力社保局2017年出站留(来)渝博士后科研项目(0108/01096101);长江师范学院引进人才科研项目(2017KYQD63);长江师范学院生物工程与现代农业专业群科研项目(CSZKY1813)。

摘　　要：以卷丹(Lilium lancifolium Thunb.)无菌小鳞片为试材,通过切片处理、优化农杆菌侵染浓度与时间以及重悬液和共培养基成分,构建农杆菌介导的高效遗传转化体系。结果表明,MS+1.5 mg·L^-16-BA+0.5 mg·L^-1 NAA+30 g·L^-1蔗糖是切片芽分化的最佳培养基,添加100 mg·L^-1抗坏血酸能有效抑制褐化并促进不定芽增殖。MS+2.0 mg·L^-1 NAA+30 g·L^-1蔗糖是生根诱导的最佳培养基。抗生素敏感性测试发现,培养基中添加Kan 100 mg·L^-1或Hyg 75 mg·L^-1结合Cef 400 mg·L^-1适宜抗性筛选。GUS染色分析表明,以去除大量元素的改良MS+100μmol·L^-1AS和去除大量元素的改良MS+1.0mg·L^-16-BA+1.0mg·L^-1NAA+100μmol·L^-1 AS为重悬液和共培养基,将切片在农杆菌菌液浓度OD600为0.4,侵染15 min,获得81.72%瞬时转化率和25.2%稳定遗传转化率。将岷江百合LrCCoAOMT转化卷丹,分子检测和GUS染色分析表明已获得转基因阳性株系。In vitro aseptic bulb scales of Lilium lancifolium Thunb.were used as the explants to establish a highly efficient Agrobacterium-mediated genetic transformation system by slicing scales,optimizing the concentration and infection time of Agrobacterium solution,adjusting the composition of the suspension solution and co-culture medium.The results showed that MS+1.5 mg·L^-16-BA+0.5 mg·L^-1 NAA+30 g·L^-1 sucrose was the optimized medium for the bud differentiation of scale slices,and adding 100 mg·L^-1 vitamin C in this medium can effectively inhibit the browning of scale slices and promote the proliferation of adventitious buds.For root induction,MS+2.0 mg·L^-1 NAA+30 g·L^-1 sucrose was the best medium choice.Antibiotic susceptibility testing revealed that the addition of 100 mg·L^-1 Kan or 75 mg·L^-1 Hyg in combination with 400 mg·L^-1 Cef in the medium was suitable for resistance screening.Taking the modified MS+100μmol·L^-1 AS as the suspension solution and the modified MS+1.0 mg·L^-16-BA+1.0 mg·L^-1 NAA+100μmol·L^-1 AS as co-culture medium,the final GUS histochemical staining indicated that the transient transformation efficiency and stable transformation efficiency can reach 81.72%and 25.2%,respectively,after inoculating scale slices in the Agrobacterium infection solution with OD600=0.4 for 15 min.Moreover,Lr CCo AOMT gene cloned from L.regale Wilson was transformed into L.lancifolium Thunb.using this genetic transformation system,and transgenic positive plants were successfully obtained based on the molecular analysis and GUS staining.

关 键 词：卷丹 遗传转化 LrCCoAOMT基因 抗性改良 

分 类 号：S682.2[农业科学—观赏园艺]