利用番茄果实特异启动子E8改造‘外源基因清除’的表达载体及转化金柑研究

Modification of‘Gene-Deletor'Vector with Tomato Fruit-specific Promoter E8 and Transformation of Kumquat

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。

In order to solve the edible safety problem of transgenic kumquat and get the non-genetic food from transgenetic crop,the foreign genes were removed from the fruit of the transgenic kumquat by using‘gene-deletor'technology which has been proven effectively in some transgenetic plants.Here,we cloned fruit specific promoter E8 from tomato and modified ‘gene-deletor'vector with E8 promoter,in which E8 promoter replace PAB5 promoter to drive recombinase FLP gene,Ca MV35 S promoter to drive report and selecting gene GUS::NPTⅡ.The seedling internode stem segments of kumquat(Fortunella crassifolia Swingle cv.Jindan) were transformed with the‘gene-deletor'vector mediated by Agrobacterium tumefaciens.We obtained 11 resistant plants and 6 transformed plants confirmed by PCR analysis.The transformed plants will be tested for the gene-deletor function later on.The improvement application of‘gene-deletor'technology in kumquat provided an alternative for citrus safety transgenic research.