利用Cre/lox特异位点重组系统获得无选择标记转AtMYB12基因的马铃薯

DEVELOPMENT OF MARKER-FREE TRANSGENIC POTATO WITH AtMYB12 GENE BY Cre/lox SITE-SPECIFIC RECOMBINATION SYSTEM

类黄酮是植物产生的一类次级代谢产物的总称,长期食用含类黄酮的果实和蔬菜有易于人体健康,目前广泛种植的马铃薯品种中仅含有痕量的类黄酮。AtMYB12是拟南芥中鉴定的调控类黄酮生物合成的特异性转录因子,并在烟草和番茄中得到了功能验证。为了增加马铃薯中类黄酮的含量,本研究构建pX6-patatin::AtMYB12无选择标记载体,利用农杆菌转化法将AtMYB12基因转入马铃薯品种Desiree,并检测选择标记NPT II基因去除以及马铃薯块茎中类黄酮积累情况。结果显示,共得到28个阳性转基因株系。随机选择其中的2个株系进行雌二醇诱导,NPT II去除效率达到8.8%,转基因马铃薯块茎中芦丁的含量最高达到3.091mg/g DW,山奈酚芸香苷含量达到0.951mg/g DW,而对照马铃薯品种Desiree中芦丁和山奈酚芸香苷等类黄酮含量低于高效液相色谱检测限值。结果表明,AtMYB12基因也能在马铃薯中调控类黄酮的合成。

As a class of secondary metabolites in plants,flavonoids is good for human health when the fruits and vegetables containing this kind of substances are often consumed.However only trace amounts of flavonoids can be detected on current widely planted potato varieties.AtMYB12 was originally identified as a flavonoids-speciflc transcription factor in Arabidopsis thaliana,which has been confirmed to activate the flavonoids synthesis in tobacco and tomato.In order to increase the content of flavonoids in potato,we constructed the marker-free vector pX6-patatin： AtMYB12,then introduced it into the susceptible cultivar Desiree via Agrobacterium-mediated stem segment transformation.The results showed that 28 positive transgenic potato lines were acquired,and the deletion efficiency of NPTII in 2 marker-free lines was up to 8.8%.The content of rutin and kaempferol rutinoside was up to 3.091 and 0.951mg/g DW in transgenic potato tubers,respectively.It is suggested that AtMYB12 can also regulate the flavonoids synthesis in potato.