利用Cre/lox重组系统建立反义基因工程雄性不育恢复系

Construct the Fertility Restoring Line of Antisense Gene Engineered Male Sterility by Cre/lox Site-specific Recombination System

利用Cre/lox重组系统中的Cre重组酶能特异性识别并介导两个同向lox位点之间DNA序列发生重组删除的特点,将TA29驱动下的反义豌豆卷须肌动蛋白基因置于两个同向lox位点之间并与Bar基因连锁,转化烟草Wisconsin 38后获得抗除草剂Basta的转基因植株。将Cre基因导入烟草Wisconsin 38建立雄性不育工程恢复系。反义Actin转基因植株与Cre转基因植株杂交获得F1,通过Cre重组酶将F1中的反义肌动蛋白基因表达盒删除实现育性的恢复。结果显示:来自豌豆卷须的肌动蛋白基因在Wisconsin 38烟草绒毡层中反义表达但未能导致明显的雄性不育,转基因植株在花器官形态、花粉形状、活力、结实、结籽等方面与野生型植株间无明显的差异。而获得的烟草Cre转基因工程恢复系除少量植株出现叶片褪绿、结果少等异常外,绝大多数植株形态结构及开花结果习性与野生型一致;其中3个Cre转基因工程恢复系与Actin反义肌动蛋白转基因植株TAA-3杂交后,杂交后代中的绝大多数反义肌动蛋白基因表达盒均被精确删除,表明将Cre/lox重组系统用于建立基于反义基因工程雄性不育的恢复系是可行的。

The Cre recombinase of the Cre/lox site-specific recombination system can catalyze deletion between two directly oriented lox sites.The antisense expression cassette of pea curl actin gene promoted by TA29 promotor flanked by two directly oriented lox sites and linked with Bar gene was transformed into Wisconsin 38 by Agrobacterium-mediated transformation,expect to obtain male sterility plants with resistance to herbicide Basta.An engineered restoring line was constructed subsequently by transformed the Cre gene into the Wisconsin 38.The F1 hybrid was obtained by cross-fertilized between antisense actin gene transgenic plants and Cre gene containing plants,the antisense actin gene expression cassette in the F1 progenies would be eliminated by Cre recombinase in the F1,which make the F1 progenies fertility.The results showed the antisense actin gene expressed in the tapetal cell of Wisconsin 38 did not bring visible male sterile phenotype.There were no obvious difference observed including flower structure,pollen shape,pollen germination rate,fruit and progenies between the antisense actin gene transgenic plants and wild-type plants.Meanwhile,including the leaf,flower and fruit of Cre transgenic plants were normal,besides few plants with phenotypes of leaf chlorophyll depigment,flower structure distortion and less fruit.F1 progenies from the cross combination of transgenic plant TAA-3 and three individual Cre transgenic plants were analyzed respectively,the results indicated the antisense actin gene expression cassette had been excised efficiently and accurately from most of F1 individuals.So,it is feasible using the Cre/lox site-specific recombination system to obtain the restoring line of male sterility resulted from antisense gene specific expression.