农杆菌介导大豆遗传转化新技术的建立及优化  

Establishment and Optimization of New Technology of A grobacterium-mediated Soybean Genetic Transformation

作　　者：石淼 韩晗 陈玥 刘涵玉 林姝含 丁晓月 闵菲 金太成[1] 杨丽萍[1] Shi Miao;Han Han;Chen Yue;Liu Hanyu;Lin Shuhan;Ding Xiaoyue;Min Fei;Jin Taicheng;Yang Liping(College of Life Sciences,Jilin Normal University,Siping,136000)

机构地区：[1]吉林师范大学生命科学学院,四平136000

出　　处：《分子植物育种》2023年第16期5295-5300,共6页Molecular Plant Breeding

基　　金：吉林省教育厅科研项目(JJKH20191013KJ);国家青年科学基金项目(31301043)共同资助。

摘　　要：农杆菌介导的植物稳定遗传转化在提高作物产量、质量及其抗逆性的分子育种中起到至关重要的作用。转基因大豆育种的遗传转化主要通过组织培养的方法获得。由于组织培养法具有实验周期长、容易污染和转化效率低的局限性,本研究在大豆(Glycine max(Linn.)Merr.)中建立并优化了一种新的快速、高效的稳定遗传转化体系,即农杆菌介导的发芽种子真空侵染法。本研究利用这种新体系完成了β-葡萄糖苷酸酶(β-glucuronidase,GUS)在大豆(Glycine max(Linn.)Merr.)品种‘沈农9号’中的表达;同时,还完成了农杆菌菌液浓度和真空侵染条件的优化。当真空侵染压强为0.08 MPa,农杆菌重悬液浓度OD_(600)为2.0时,稳定遗传转化效率达到10%。GUS化学组织染色检测结果表明,转基因T_(1)代中获得16个GUS转基因大豆植株。半定量RT-PCR结果表明,在T_(2)代转基因植株中GUS基因获得了稳定表达。本研究建立的体系具有快速、操作简单和遗传转化效率较高的优势,为大豆稳定遗传转化研究提供了新的技术方法。Agrobacterium-mediated stable plant genetic transformation plays an important role in molecular breeding to improve crop yield,quality and stress resistance.Stable transformation of genetic soybean breeding is mainly obtained by using tissue culture.Due to the long time of experiment,easy to pollute and low efficiency limitation of tissue culture,we established and optimized a new rapid and high efficience system of stable genetic transformation in soybean(Glycine max(Linn.)Merr.)plants,namely A grobacterium-mediated vacuum inoculation of germinated seeds.The expression of β-glucuronidase(GUS)was performed in soybean variety‘Shennong No.9’by using this new system.Moreover,we performed optimization of A grobacterium concentration and vacuum inoculation conditions.The efficiency of stable genetic transformation reached 10% when the vacuum pressure was 0.08 MPa and the Agrobacterium resuspension concentration OD_(600)was 2.0.The results of GUS chemical tissue staining showed that 16 GUS transgenic soybean plants were obtained in transgenic plants of T_(1)generation.The results of semi-quantitative RT-PCR showed that GUS gene was stably expressed in the transgenic plants of T_(2)generation.The system established in this study has the advantages of fast and simple work,and higher efficiency of genetic transformation,which provides a new technical system for soybean stable genetic transformation.

关 键 词：大豆 稳定遗传转化 发芽种子真空侵染法 β-葡萄糖苷酸酶 

分 类 号：S565.1[农业科学—作物学]