外源脱水应答转录因子CBF4基因转化玉米的研究

【作者】 杨东歌；

【导师】 张晓东；

【作者基本信息】 首都师范大学 ， 植物学， 2009， 硕士

【摘要】 玉米是世界上三大粮食作物之一,也是重要的饲料作物,在世界农业生产中占有相当重要的地位。在我国,玉米的总产已经超过小麦,成为第二大粮食作物。但是,我国每年约有50%以上的玉米种植在干旱和半干旱地区。干旱是限制我国玉米生产发展和产量提高的第一要素。因此,培育抗旱玉米品种非常重要。以杂种优势利用为核心的传统育种方法在提高玉米产量上已取得了巨大成功。然而,玉米的抗旱性状属于数量性状,由于传统育种方法的局限性,已经不能满足人们越来越高的需求。近年来,利用转基因技术提高玉米抗旱能力的研究受到世界各国科学家的广泛关注。目前,利用基因工程技术培育抗旱品种主要有以下几种策略,包括转渗透调节物质合成关键酶基因、转活性氧清除酶类基因、转抗旱基因和转编码转录因子的基因等。转录因子由于能激活下游一系列抗逆基因的表达而逐渐受到人们的重视,转转录因子抗旱基因工程与转单个抗旱基因相比具有更大的优越性。为了提高玉米的抗旱能力,加速我国玉米抗旱育种的进程,本研究克隆了拟南芥脱水应答转录因子CBF4基因并将其转化到玉米中去,以期得到抗旱能力有所提高的转基因玉米株系,主要取得以下进展:1)克隆了拟南芥CBF4基因和逆境诱导表达基因rd29A的启动子,以草甘膦为筛选标记基因,构建了rd29A启动子驱动下的CBF4基因逆境诱导表达载体pBAC146;2)分别用基因枪法和花粉管通道法将pBAC146载体转化到玉米优良自交系中,其中基因枪转化法获得36株转化植株,花粉管通道法收获了1270粒转化种子;3)基因枪法获得的T₀代转基因植株经PCR检测大部分呈阳性,南繁后4株转基因植株成功结实,对T₁代转基因株系的PCR、PCR-Southern、Southern杂交检测表明,3个转基因株系成功整合了外源目的基因CBF4;4)为检测所获得的转基因植株的抗旱能力,将T₁代转基因植株收获的种子（T₂代）种植到温室花盆中进行人工抗旱处理后,对其抗旱相关生理指标,包括相对含水量,叶绿素含量和脯氨酸含量等进行检测,各项指标测定结果均表明,转基因植株的抗旱能力相比对照有所提高;5)由于花粉管通道法获得的种子较多,为进行快速有效地筛选,进行了草甘膦预实验,筛选出了合适的草甘膦浓度,为下一步阳性植株的筛选奠定了基础。本研究表明,利用脱水应答转录因子CBF4基因在转基因玉米中的诱导表达,可以提高玉米的抗旱能力,我们的工作为进一步为利用CBF4基因进行玉米抗旱育种奠定了基础。更多还原

【Abstract】 Maize,one of the three important cereal crops in the world,plays a very important role in food and animal feed production.As its whole production surpassed wheat,maize has become the second largest cereal crops in china.More than fifty percent of maize planted in arid and semiarid region every year.And through the whole growth stage of maize,different degrees of damage to its growth might be caused by drought and less of water,which are the main factors restricting maize production.So,it is very important to breed drought resistant maize.Traditional breeding way using heterosis as a main method has made a great success on improvement of maize production.As quantitative character of drought resistance, traditional breeding has too many limitations to satisfy the needing of breeders in the future. In recent years,to improve drought resistance of maize by transgenic method has got great attention in many countries.There are several kinds of gene we can use in breeding stress resistant plant,Genes for osmotic regulation substance synthesis,for example,genes coding for enzymes for active oxygen clearing,genes coding for proteins induced by abiotics or genes acts in the stress signal transduction pathway.In order to gain high drought resistance maize and speed up the process of drought resistance breeding,the dehydration responsive transcriptional factor CBF4 gene from Arabidopsis thaliana was cloned and transformed into maize with biolistic bombardment.The main results are as follows:1) The CBF4 gene and the promoter of rd29A gene,a preceding drought responsive gene, were amplified from Arabidopsis thaliana by PCR.Using the EPSP gene as the selective marker,pBAC146,the expression vector of CBF4 gene drived by the promoter of rd29A gene was constructed.2) Biolistic bombardment and pollen-tube pathway were used to transform the pBAC146 vector into excellent maize inbred lines.Thirty six transformation plants transformed by Biolistic bombardment were obtained and 1270 seeds were harvested through the pollen-tube pathway.3) PCR analysis suggested that the transgene exist in most of the 36 transformation plants, among which four plants have successfully gotten seeds.PCR,PCR-Southern and Southern analysis proved that the alien CBF4 gene had been integrated into the genome of 3 transgenic lines.4) To test the drought resistence ability of the transgenic lines we got,seeds of T1 generation （T₂ generation） were planted into garden pot in greenhouse and treated by no watering. The relative water content,proline content and chlorophyll content analysis showed that transgenic lines had higher drought resistant ability than non-transgenic plants.5) To select those seeds obtained by pollen tube pathway quickly and effectively,normal maize seeds were treated by different content of glyphosate.The suitable glyphosate content for distinguishing transgenic and non-transgenic plants was found.Thisr study suggested that induced expression of the CBF4 gene in transgenic maize could improve the drought resistance of transgenic maize,and which will pave a way for using CBF4 gene in maize drought resistant breeding.更多还原