摘要
Cre loxP是来源于噬菌体P1的一种位点特异性重组系统。目前 ,它已广泛应用于基因功能鉴定 ,动植物及微生物基因组的修饰以及基因的表达和调控。雄性不育系在杂种优势的利用中具有重要意义。本实验是以Cre loxP系统调控细胞毒素基因barnase在植物雄性器官发育的特异时期在特异组织表达 ,从而达到控制植物育性的目的。1 在本实验中 ,通过PCR方法克隆了解淀粉芽孢杆菌Bacillusamyloliquefaciens的barnase基因 ,及其抑制因子barstar的DNA序列 ,同时 ,构建成功双元表达载体及基因枪转化载体 ,并已获得经分子检测呈阳性的小麦植株。2 osg6B是来源于水稻花药绒毡层的特异性的启动子 ,它在小孢子发育的四分体时期至单核花粉期在花药绒毡层中特异启动基因的表达 ,它具有较为严紧的时空特异性。以osg6B为启动子 ,构建了一组受控于Cre/loxP位点特异性重组系统的适用于基因枪法转化小麦的载体系统。其中 ,转不育基因(og6B barnase)小麦植株生长正常 ,但开花后 ,花粉量明显减少 ,自交不能得到正常种子。进行花粉活力检测发现 ,转基因小麦大部分花粉表现为败育。花粉离体萌发试验表明 ,转不育基因小麦的花粉离体萌发率极低 ,几近败育。说明以osg6B驱动barnase在小麦花药中的表达可以导致雄性不育。3
Cre/loxP is a site specific recombination system from phage P1. Presently, it has been extensively used in the identification of gene function, genome modification, gene expression and gene regulation in plants, animals and microorganisms. Male sterility is an important character in the utilization of hybrid heterosis. This work was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male apparatus under the control of Cre/loxP system. 1 In this study, we cloned barnase gene and it is strong inhibitor from Bacillus amyloliquefaciens . We successfully constructed a series of plasmids suitable for biolistic and Agrobacterium tumefacien mediated transformation respectively. Positive transgenic plants were obtained by molecular identification. 2 osg6B, a promoter from rice, can direct gene expressing in anther tapetal during the early developmental stage of microspore from tetrad to uninucleate pollen, with high temporal and spatial specificity. Combining the osg6B promoter and Cre/loxP system, we constructed a set of plasmids suitable for biolistic transformation. We observed that wheat transformed with sterile gene grown normally, but the amount of pollen decreased significantly after flowering. Consequently, it was hard to produce normal selfed seeds. Pollen vigor testing showed that most of the pollen of transgenic wheat were sterile. In vitro germination rates of pollen obtained from transgenic wheat were same result, indicating that barnase gene under the control of osg6B can results in male sterility. 3 we also constructed a set of plasmids containing Cre/loxP system driven by the osg6B and transformed tobacco via the mediation of Agrobacterium tumefacien. The tobacco integrated with Cre gene was transformed for the second time with binary vector harboring blocking fragment via Agrobacterium tumefacien. Although the vegetative growth of transgenic tobacco followed second transformation was normal, the reproductive organ showed many metamorphosis such as petal spliting abnormally or without spliting, filaments shortening or adhering together, anther spliting abnormally or wilting ealier than normal time. These phenomena were probably caused by the specificity of the promoter or by the leaky expression of barnase gene. 4 The staining of pollen harvested from the secondly transformed lines showed different fertility from whole sterility, partial sterility to most fertility, and similar results were obtained from in vitro pollen germination experiments. This demonstrated that, using Cre/loxP system, sterile lines can be obtained through selecting transgenic plants. 5 In cloning of barnase gene, we obtained 9 mutants. By analysing these sequences, we deduced that the 14th aa and 80th aa of barnase are important for its activity. Meanwhile, we found that the 316th base is susceptible to mutation and sometimes inserted by an additional C, resulting in the change of the stop codon. These phenomena may provide useful imformation for furture study on barnase gene.
出处
《分子植物育种》
CAS
CSCD
2003年第4期557-558,556,共3页
Molecular Plant Breeding
基金
国家转基因植物研究与产业化专项,课题类别及编号为:J2000-B-021。
