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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 highly efficient transgenic system was established with hypocotyl hypocotyls as explants. Using this method, a fusion expression vector pCAMBIAI35S-2300-SmARF8-INT with the NPTⅡ gene as the screening marker was constructed for the purpose of obtaining RNA interference transgenic plants of SmARF8 gene in eggplant. Implant. MS selection medium containing 2 mg · L -1 indole acetic acid, 0.5 mg · L -1 zeatin, 25 mg · L -1 kanamycin and 500 mg · L -1 cephalexin directly induced explants Produce transgenic seedlings, the regeneration rate of 80%. Transgenic shoots continue to grow and produce robust roots on hormone-free MS medium, ultimately yielding transgenic lines. RT-PCR and qRT-PCR analysis confirmed that the expression of SmARF8 in T0 transgenic lines was not expressed or decreased after interference. Southern blot analysis showed that there was one T-DNA inserted copy in the selected T0 transgenic lines . The above results show that the use of the established transgenic system SmARF8 gene was successfully knocked out in eggplant.