刺葡萄DFR基因植物表达载体构建及转化茉莉花愈伤组织的研究

Plant Expression Vector Construction and Jasmine(Jasminum sambac(Linn.) Aiton) Callus Transformation of DFR Gene from Spine Grape(Vitis davidii Fo?x.)

本研究以从刺葡萄克隆获得的DFR基因为目的基因,同时从质粒p CAMBIA1302中扩增获得Ca MV35S启动子和NOS终止子,并利用双酶切法构建由Ca MV35S启动子驱动DFR基因且带有NOS终止子的表达载体,验证测序结果显示成功构建了p CAMBIA1302-35S-DFR-NOS植物表达载体,并将该载体转化到农杆菌EHA105中。采用农杆菌注射渗透法转化烟草叶片进行瞬时表达,经鉴定,转化后的烟草叶片中能检测到GFP基因的表达。采用农杆菌介导法将该植物表达载体转化受体茉莉花愈伤组织,经多轮抗性筛选获得了转基因抗性愈伤组织,经PCR鉴定,茉莉花转基因抗性愈伤组织中检测到GFP、Hyg和DFR基因等的存在,初步证明目的基因DFR已成功整合到宿主的基因组中。茉莉花愈伤组织遗传转化体系的建立,验证了花色苷合成相关基因转化茉莉花愈伤组织的可行性,为今后通过植物转基因技术丰富茉莉花的花色奠定基础。

In this experiment, DFR gene encoding dihydroflavonol 4-reductase obtained from spine grape（Vitis davidii Fo？x.） was taken as the target gene, meanwhile, the promoter Ca MV35 S and terminator NOS was amplified from plasmid p CAMBIA1302. Then, the plant expression vector, which inserted into DFR gene driven by promoter Ca MV35 S and combined with terminator NOS, was constructed by using double enzyme digestion technique. The detecting and sequencing results revealed that the plant expression vector p CAMBIA1302-35 S-DFR-NOS was constructed successfully. After that the vector was introduced to Agrobacterium tumefaciens EHA105. The transient expression of GFP gene in tobacco leaves was checked with A. tumefaciens EHA105 mediated transformation, and it turned out the GFP gene could be expressed. The plant expression vector p CAMBIA1302-35 S-DFR-NOS was introduced to jasmine calli using agrobacterium-mediated transformation. And then, the resistance jasmine calli was obtained after repeatedly resistance screening. GFP, Hyg and DFR were identified to exist in the genome of resistance jasmine calli by PCR. The preliminary evidence showed that the exogenous DFR gene was integrated into the host cell genome in jasmine calli. The feasibility of the genes related to anthocyanins synthesis introduced into jasmine genome was verified through the establishment of genetic transformation system in jasmine. This study would help build a foundation to enrich jasmine flower colors using plant transgenic technology.