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利用“外源基因清除”技术(‘Gene-Deletor’),将外源基因从转基因金柑果实中彻底清除,可以达到用转基因作物生产出非转基因食品的目的。本研究利用引物pE8F和pE8R从樱桃番茄中克隆果实特异启动子E8,通过SalⅠ和SmaⅠ双酶切置换掉“外源基因清除”载体(pCambia-LF-polseed-FLP,简称Y-A)中的花粉、种子特异启动子PAB5,重新构造“外源基因清除”载体Y-A-E8,利用农杆菌EHA105介导转化实生态金柑,获得转化植株,并通过nptⅡ基因特异引物nptⅡ-F和nptⅡ-R PCR检测阳性转化植株。载体Y-A-E8 SalⅠ和SmaⅠ双酶切结果表明,E8启动子成功替换启动子PAB5,Y-A-E8经EHA105介导转化成功获得11株转化植株且PCR检测到6株为Y-A-E8转基因植株。研究结果可为“外源基因清除”技术在转基因金柑方面的应用提供参考。
The purpose of using transgenic crops to produce non-genetically modified foods can be achieved by using “Gene-Deletor” to completely remove exogenous genes from the fruits of the transgenic kumquats. In this study, we used the primers pE8F and pE8R to clone the fruit-specific promoter E8 from cherry tomato and double-digested with SalI and SmaI to replace the “pCambia-LF-polseed-FLP” Pollen and seed-specific promoter PAB5 was recombined to construct “exogenous gene clearance” vector YA-E8. Agrobacterium tumefaciens EHA105 was used to transform and transform the wild kumquat, and the transformed plants were obtained. The nptⅡ-F and nptⅡ- R PCR detection of positive transformed plants. The results of double enzyme digestion with SalⅠ and SmaⅠ of vector Y-A-E8 showed that the E8 promoter successfully replaced the promoter PAB5 and Y-A-E8 was successfully transformed into EHA105. Eleven transformed plants were obtained and 6 Y-A-E8 transgenic plants were detected by PCR. The results can provide a reference for the application of “exogenous gene clearance” technology in transgenic kumquat.