【作者】 高宜峰；

【导师】 张金文；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2013， 硕士

【摘要】 人参果是以营养器官繁殖为主的多年生草本植物，连年种植容易造成多种病毒病积累。为了培育同时抵御多种病毒病的人参果转基因植株，本试验以危害人参果的主要病毒烟草花叶病毒（TMV）、马铃薯M病毒和黄瓜花叶病毒（CMV）为对象，筛选靶标序列，用重叠延伸PCR方法将三个靶标片段融合在一起，构建了3种病毒基因靶标片段融合基因的植物ihpRNAi载体。1. TMV、PVM和CMV靶标片段的选择与克隆。根据GenBank中烟草花叶病毒（TMV）P126基因[GI:371786069]、马铃薯M病毒（PVM）CP基因[GI:361071296]和黄瓜花叶病毒（CMV）2b蛋白基因[GI:83282715]序列，通过E-RNAi网站在线分析，筛选出TMV P126基因片段、PVM CP基因片段和CMV2b蛋白基因片段，这些片段含有较多有效小干扰RNA[siRNA，19nt]的长dsRNA片段，可作为构建干扰载体的目的片段。2.靶标片段的融合及反向重复表达载体的构建。采用重叠延伸PCR技术（splicing-by-overlap extension PCR，SOE-PCR），首先构建融合基因PC和TPC（正向插入片段F），再扩增反向插入片段R。然后将正向片段TPC（F）和反向片段分别插入载体pHANNIBAL所含内含子（intron）两侧，获得重组质粒pHAIFR，再将含intron的正反向片段整体酶切后插入载体pCEPSPS，最终得到含融合基因TPC反向重复的植物表达载体pCEIHFR。3.人参果的遗传转化。将pCEIHFR通过冻融法导入农杆菌LBA4404，采用叶盘法转化人参果，获得了人参果再生植株，为后续实验奠定了基础。并验证了人参果叶盘法遗传转化体系中最佳的激素组合为ZT和IAA。更多还原

【Abstract】 Pepino(Solanum muricatum Ait.) is a perennial herb with vegetative propagation. It iseasy to be infected by many viruses due to continuous planting. In order to find a newmethod for defending viral diseases, the purpose of this experiment was to study three kindsof harmful viruses which were common in pepino. The RNAi target sequences that containsthe more effective small interfering RNA[siRNA, long19nt] fragments were filtered basedon the genes sequence of Tobacco mosaic virus (TMV) P126, potato virus M (PVM) CP andCucumber mosaic virus (CMV)2b in GenBank. The splicing-by-overlap extension PCR wasused to fuse three target sequences in together, and at the same time to construct a invertedrepetitive structure with pdk (pyruvate orthophosphate dikinase) intro of plant RNAiexpression vector pCEIHFR which was supposed to form intron splicing hpRNA (ihpRNA)after transcription.1. Selecting and cloning the target fragments from the gene sequence of TMV、PVMand CMV. Importing respectively the GenInfo Identifier is371786069、361071296and83282715into E-RNAi website to filter the target segments. These fragments contain moreeffective small interfering RNAs which can be used of building interference vector fragment.2. Fusion of target fragments and construction of expression vector. Using thetechnology of splicing-by-overlap extension PCR to building the fusion gene PC and TPCone after another, then amplification the reverse insertion fragment R. Then both the sensefragment TPC(F) and the antisense fragment CPT(R) were inserted into the two sides ofintron of vector pHANNIBAL in priority order, and the recombinant plasmid pHAIFR wasconstructed. The sense and antisense fragments with intron were further inserted into thevector pCEPSPS, and the recombinant vector pCEIHFR was acquired ultimately.3. Transformation of Pepino (Solanum muricatum). The recombinant plant-expressionvector pCEIHFR was introduced into Agrobacterium tumefaciens LBA4404by directlytransferring. Finally, the regenerated pepino plants were obtained and laying the foundationfor subsequent experiments.更多还原