紫红线茄转基因体系优化及SmARF8基因RNA干涉表达载体的遗传转化

Optimization of the Transgenic System and Genetic Transformation of SmARF8 Gene RNAi Vector in Red-purple Long Eggplant

建立了以紫红线茄下胚轴为外植体的高效转基因体系。利用该方法,以获得茄子SmARF8基因RNA干涉转基因植株为目的,构建了以NPTⅡ基因为筛选标记的融合表达载体pCAMBIAI35S-2300-SmARF8-INT,并通过农杆菌介导,侵染茄子下胚轴外植体。含有2 mg·L-1吲哚乙酸,0.5 mg·L-1玉米素,25 mg·L-1卡那霉素和500 mg·L-1头孢氨苄的MS选择培养基可直接诱导外植体产生转基因分化苗,再生率达到80%。转基因苗继续生长并在不含激素的MS培养基上产生健壮根系,最终获得转基因株系。RT-PCR和qRT-PCR分析验证了T0代转基因株系中内源基因SmARF8被干涉后不表达或表达量降低,Southern blot分析显示选择的T0代转基因株系中存在1个T-DNA插入拷贝。以上研究结果表明利用建立的转基因体系SmARF8基因在茄子中被成功敲除。

A high efficient genetic transformation system was developed using hypocotyl explants for producing transgenic red-purple long eggplant(Solanum melongena L.). To generate SmARF8 gene RNAi transgenic eggplant plants,the binary vector pCAMBIAI 35S-2300-SmARF8-INT with the selectable marker gene neomycin phosphotransferase(NPTⅡ)was constructed and transformed into eggplant hypocotyl explants via Agrobacterium tumefaciens-mediated infection. Transgenic plantlets were induced on MS selective media containing 2 mg · L-1 indole-3-acetic acid,0.5 mg · L-1 zeatin,25 mg · L-1 kanamycin and 500 mg · L-1 cefotaxime with regeneration ratio higher to 80%. Subsequently,the transgenic plantlets kept growing on same medium and then rooted efficiently on MS basic media to become transgenic plants. The RT-PCR and qRT-PCR analysis showed no or less transcript of SmARF8 in T0 transgenic generation. Further Southern blot analysis of selected T0 transgenic plants confirmed the presence of single T-DNA insertion. These results demonstrate that the endogenesis SmARF8 has been successfully knocked out in eggplant using the established transgenic system in this study.