矮牵牛转基因体系的建立及转BIO和bio基因研究初报  

Establishment of genetic transformation system and initial report of transform BIO and bio gene in Petunia hybrida

作　　者：李颖[1] 刘姬艳[1] 胡江琴[1] 陈哲皓[1] 胡灵芝[1] 王利琳[1] 

机构地区：[1]杭州师范大学生命与环境科学学院,杭州310036

出　　处：《浙江大学学报（农业与生命科学版）》2013年第1期42-49,共8页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江省自然科学基金资助项目(Y3100273;Y3100241)

摘　　要：BIO ORGANS(BIO)是百脉根中调控器官形态及大小的基因。首先改良了矮牵牛愈伤诱导和再生体系,并进一步利用根癌农杆菌介导法定向将BIO及其突变基因bio导入矮牵牛中进行功能研究。结果证实含0.1mg/L6-BA的MS培养基有利于矮牵牛组培苗的增殖,添加了1.0mg/L 6-BA和0.1mg/L NAA的MS培养基有利于愈伤以及不定芽的诱导。对转基因植株目的基因的PCR检测结果显示,BIO及其突变基因bio被成功转入了矮牵牛中。表型观察结果显示:转BIO及其突变基因bio的植株叶片边缘均呈现不规则形态,部分叶片缺刻,出现由一个叶片向两个叶片分裂的趋势。其中转BIO基因植株部分叶片面积减小,叶片变窄或几乎只剩主叶脉。该研究证实BIO基因对保持叶片器官形态起到关键的作用,从而为进一步探索BIO基因的调控机制提供了实验依据。Summary BIO ORGANS （BIO） gene is located on the short arm of Chromosome IV in Lotusjaponicus. The bio mutant is obtained from a large scale EMS mutagenesis screen. Gene sequencing have proved that a stop coden appeared earlier in the transcripts of BIO by a retrotransposon insertion, which led to a truncated protein. Morphological analysis showed that the bio mutant displayed enlarged plant organs and a better symmetry in flower. It has been speculated that LjBIO regulate organ morphology and plant size in Lotus japonicus. However, the function and regulation mechanism of LjBIO still remains unclear. Petunia hybrida is a good material for genetic engineering and molecular biology research, due to its short life cycle, simple growing conditions and mature transgenic system mediated by agrobacterium infiltration. Calli tissues are induced from P. hybrida QL01 explants as transgenic materials in order to gain further information about the function and possible mechanism of LjBIO. The tissue culture and regeneration system of P. hybrid was refined. Different explants from P. hybrida QL01 were chosen and several concentration gradients of 6-BA and NAA were tested for selecting the best tissue cultureand regeneration system. Agrobacterium mediated gene transformation was used to induce BIO and the mutant gene bio into the calli of QL01. The 35S-BIOGFP and 35S-bio-GFP vectors were transferred into agrohacterium GV3101 by freezing and defreezing immediately using nitrogen and water bath. The target agrobacteria clones were verified by PCR and were cultured overnight into large volume. The agrobaeterium were resuspended in MS liquid medium, used to infiltrate the pre-cultured explants for 8 minutes and co-cultured for another 3 days in dark. Transgenic plants were obtained and confirmed with hygromycin screening and RT PCR detection. The phenotypes of transgenie plants were recorded and analyzed to obtain more information about the gene function. Results showed that MS medium with 0. 1 mg/L 6-BA was suitable for th

关 键 词：矮牵牛 BIO ORGANS基因(BIO) 组织培养 遗传转化 

分 类 号：Q78[生物学—分子生物学]