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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (1): 42-    DOI: 10.3785/j.issn.1008-9209.2012.04.051
生物科学与技术     
矮牵牛转基因体系的建立及转BIO, bio基因研究初报
李颖， 刘姬艳， 胡江琴， 陈哲皓， 胡灵芝， 王利琳*
(杭州师范大学 生命与环境科学学院，杭州 310036)
Establishment of genetic transformation system and initial report of transform BIO, bio gene in Petunia hybrida
LI Ying，LIU Jiyan，HU Jiangqin，CHEN Zhehao, HU Lingzhi，WANG Lilin*
(
College of Life and Environment Sciences, Hangzhou Normal University, Hangzhou 310036,China
)
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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0.1 mg/L 6-BA 的MS培养基有利于矮牵牛组培苗的增殖，添加了1.0 mg/L 6-BA和0.1 mg/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基因的调控机理提供了实验依据。
Abstract: The BIO organs (BIO)  gene is located on the short arm of Chromosome Ⅳ in Lotus japonicus. 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 culture and regeneration system. Agrobacterium mediated gene transformation was used to induce BIO and the mutant gene bio into the calli of  QL01. The 35SBIOGFP and 35SbioGFP vectors were transferred into agrobacterium 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 agrobacterium 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 transgenic 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 the growth of P. hybrid and the proliferation of calli from the excised stem. MS medium with 10 mg/L 6BA and 01 mg/L NAA was best for the bud regeneration. 03 mg/L hygromycin was used for resistance screening and 20 different resistant plants were obtained for both BIO and its mutant gene bio respectively. PCR detection of gene expression showed  that 15 35SBIO and 17 35Sbio  transgenic plants had    been successfully obtained. Morphological analysis displayed  that both 35SBIO and 35Sbio transgenic plants had  abnormal leaves with irregular margins,  some of them even showed  a tendency that one leaf would be divided into two. In 35SBIO transgenic plants, certain blades had  smaller size and became thinner than the wildtype and a  few of them only had main veins left. Detailed data showed that leaf surface area and lengthwidth ratio altered  obviously. The ectopic and overexpression of LjBIO and Ljbio in P. hybrida revealed  that this gene affected  not only the size of plant and the symmetry of floral organs, but also influenced  the development and morphology of leaf. The study illustrated  the essential role of LjBIO gene in maintaining leaf morphology, and displayed that  the 3′ end of LjBIO had  its unique function  worthy of further research according to the different leaf phenotypes between 35SBIO and 35Sbio transgenic plants. This work also provided experimental basis for the further study of regulatory mechanism of LjBIO gene.  
出版日期: 2013-01-20
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李颖
刘姬艳
胡江琴
陈哲皓
胡灵芝
王利琳*

引用本文:

李颖， 刘姬艳， 胡江琴， 陈哲皓， 胡灵芝， 王利琳*. 矮牵牛转基因体系的建立及转BIO, bio基因研究初报[J]. 浙江大学学报(农业与生命科学版), 2013, 39(1): 42-.

LI Ying，LIU Jiyan，HU Jiangqin，CHEN Zhehao, HU Lingzhi，WANG Lilin*. Establishment of genetic transformation system and initial report of transform BIO, bio gene in Petunia hybrida. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(1): 42-.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.04.051    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I1/42

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