转Barnase基因无籽植物的研究  

Developing Transgenic Plants with No Seeds by Transforming Barnase Gene

作　　者：陈桂敏[1] 吴刚[1] 武玉花[1] 卢长明[1] 

机构地区：[1]中国农业科学院油料作物研究所,农业部油料作物生物学与遗传育种重点实验室,武汉430062

出　　处：《农业生物技术学报》2012年第2期113-120,共8页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2008AA10Z152)

摘　　要：种子败育的无籽瓜果具有较强的市场竞争力和可观的经济价值。来源于解淀粉芽胞杆菌(Bacillus Amyloliquefaciens)的Barnase基因编码12kD的胞外小分子核糖核酸酶(RNAase)。在缺乏其天然抑制物Barstar的情况下,单独表达Barnase通常会造成宿主细胞的死亡。本研究用油菜(Brassicanapus)种子特异启动子Napin调控Barnase基因,构建T-DNA表达载体,并用叶盘转化法转化烟草(Nicotiana benthamiana),获得55株转基因植株。随机抽取10株对其花器官进行形态学分析,并比较转基因植株和非转基因植株在不同发育时期花器官的形态学变化,发现转基因植株和非转基因植株在授粉前的花器官形态没有明显差异,但是受精以后转基因植株花瓣逐渐枯萎,不能正常发育形成种子,而非转基因植株能够正常形成种子。为了进一步了解转基因烟草种子败育的原因,将转基因植株和非转基因植株正反杂交,均不能正常结籽,表明转基因烟草的种子败育不是由自交不亲和性引起的。将转基因植株和非转基因植株花粉分别使用亚历山大染液染色,显微观察花粉的大小、形状和色泽,发现转基因花粉和非转基因花粉无明显差别,转基因植株和非转基因植株均能产生具有正常授粉活性的花粉。以上研究表明,使用种子特异性启动子驱动Barnase基因表达,外源基因不会通过花粉渗漏表达。本研究成功获得了Barnase基因在种子中特异表达的无籽转基因烟草,该研究为无籽植物的研制提供了一种全新的模式和思路。Seedless fruit has strong market competitiveness and the considerable economic value,seedless trees can effectively reduce the pollution of the environment,seedless transgenic plants can help prevent foreign gene dispersal.Barnase gene comes from Bacillus amyloliquefaciens,encoding a 12 kD small molecule extracellular ribonuclease（RNAase）.In the absence of its natural inhibitor Barstar,the expression of Barnase gene usually causes the death of its host cell.The T-DNA vector i.e.pBIloxP2 PNap TAibarnase carrying Barnase gene driven by Brassica napus seed-specific promoter Napin was constructed and transformed into tobacco（Nicotiana benthamiana） with the leaf disc transformation method,and 55 tobacco transformants were obtained.In which,10 transformants were randomly selected for floral morphology analysis.Comparison of floral organs indicated that no obvious morphological difference was observed at different developmental stages between the transgenic and non-transgenic plants before pollination.Whereas,the floral organs of transgenic plants were gradually shrinking and dry,and failing to produce seeds after pollination;the petals of non-transgenic tobaccos were normally withered and can produce seeds within torus after pollination.In order to understand the mechanism of seed abortion of transgenic tobaccos,the cross between transgenic and non-transgenic plants were performed.However,no seed was obtained with the transgenic plants either as female parent or male parent.The hybrid results revealed that the seed abortion of transgenic tobacco was not caused by the self-incompatibility.To analyze the pollen activity,Alexander solution was used to stain the pollens of transgenic and non-transgenic tobaccos,then we observed the size,shape and color of pollens from transgenic and non-transgenic tobaccos with microscope,no difference was found.Therefore both the transgenic and non-transgenic tobaccos could generate active pollens.It is concluded that the seed specific promoter Napin used in this study only drives

关 键 词：Napin启动子 Barnase基因 转基因烟草 无籽 

分 类 号：S792.110.4[农业科学—林木遗传育种]