[1]孙式静,杨素欣,冯献忠.超声波辅助处理农杆菌介导大豆胚尖转化转基因植株的获得和分子鉴定[J].大豆科学,2014,33(06):808-814.[doi:10.11861/j.issn.1000-9841.2014.06.0808]
　SUN Shi-jing,YANG Su-xin,FENG Xian-zhong.Molecular Characterization of the Transgenic Soybean Plants by Sonication-assisted Agrobacterium-mediated Transformation Using the Embryonic Tips[J].Soybean Science,2014,33(06):808-814.[doi:10.11861/j.issn.1000-9841.2014.06.0808]
点击复制

超声波辅助处理农杆菌介导大豆胚尖转化转基因植株的获得和分子鉴定

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第33卷 期数: 2014年06期 页码: 808-814 栏目: 出版日期: 2014-12-25
Title:
Molecular Characterization of the Transgenic Soybean Plants by Sonication-assisted Agrobacterium-mediated Transformation Using the Embryonic Tips
文章编号:
1000-9841.2014.06.0808
作者:
孙式静1; 杨素欣1; 2; 冯献忠1; 2
1.山东省高校系统生物学重点实验室/山东师范大学 生命科学学院,山东 济南 250014；
2.中国科学院 东北地理与农业生态研究所/中国科学院 大豆分子设计育种重点实验室，吉林 长春 130102
Author(s):
SUN Shi-jing1; YANG Su-xin1; 2; FENG Xian-zhong1; 2
1.College of Life Science,Shandong Normal University/Key Laboratory of Systems Biology in Universities of Shandong,Jinan 250014,China;
2.Key Laboratory of Soybean Molecular Design Breeding,Northeast Institute of Geography and Agroecology,Chinese Academy of Sciences,Changchun 130102,China
关键词:
大豆; 胚尖; 农杆菌转化; 超声波辅助处理
Keywords:
Glycine max; Embryonic tips; Agrobacterium-mediated transformation; Sonication-assisted treatment
分类号:
S565.1
DOI:
10.11861/j.issn.1000-9841.2014.06.0808
文献标志码:
A
摘要:
以大豆Williams 82成熟胚尖为试验材料，建立了超声波辅助处理的大豆胚尖农杆菌转化体系，研究了不同超声波处理时间对报告基因UidA(GUS）瞬间表达的影响和不同草铵膦浓度对大豆转化效率的影响。结果表明：在超声波处理2 min时，报告基因GUS的瞬时表达效率达到最高；在0.3～0.6 mg.L-1草铵膦梯度筛选浓度下，阳性转基因植株的数量最多。对于所获得的转基因株系进行了PCR检测和除草剂Basta涂抹抗性检测；实时荧光定量PCR检测显示转基因植株的目的基因的表达量达到野生型的1.7～42倍,Southern 杂交检测表明外源基因已经整合到大豆基因组中。
Abstract:
Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing.Despite the popularity of the method,low efficiency of soybean transformation is a major challenge for scientists.Modification of transformation method may lead to better understanding of the system and result in high efficiency transformation.In this paper,the sonication-assisted Agrobacterium-mediated transformation(SAAT)system of soybean(Glycine max)was set up using embryonic tips of soybean cultivar of Williams 82.The effects of different SAAT duration and glufosinate concentration on transformation efficiency were investigted.The results showed that the transient expression of UidA(GUS)reporter gene reached the hightest with 2 minutes sonication treament among various time durations.The gradient seletion of glufosinate concentration from 0.3 to 0.6 mg.L-1produced the highest transformation rate.Transgenic plants were verified by PCR analysis and herbicide paint assay.Real-Time Quantitative PCR showed the expression of target genes in transgenic plant was about 1.7 to 42 times than that in wild type.Southern blot result proved the target gene was integrated into the soybean genome of transgenic plants.This research will provide new information for soybean transformation improvement.

参考文献/References:

［1］于洋,侯文胜,韩天富.农杆菌介导大豆遗传转化技术的研究进展 ［J］.大豆科学，2010，29(4):696-701.(Yu Y,Hou W S,Han T F,et al.Approaches to Agrobacterium-mediated transformation in soybean［J］.Soybean Science,2010,29(4):696-701.)

[2]杨向东,隋丽,李启云,等.大豆遗传转化技术研究进展［J］.大豆科学，2012，31(2):302-315.(Yang X D,Sui L,Li Q Y,et al.Recent advances in soybean transformation［J］.Soybean Science,2012,31(2):302-315.)
[3]Trick H N,Finer J J.Sonicaton-assisted Agrobacterium-mediated transformation of soybean[Glycine max(L.)Merrill] embryogenic suspension culture tissue［J］.Plant Cell Reports,1998,17(6-7):482-488.
[4]Santarem E R,Trick H N,Essig J S,et al.Sonication-assisted Agrobocterium-mediated transformation of soybean immature cotyledons:optimization of transient expression［J］.Plant Cell Reports,1998,17(10):752-759.
[5]刘海坤,卫志明.一种大豆成熟种子的消毒方法［J］.植物生理学通讯,2002,38(3):260-261.(Liu H K,Wei Z M.A method for sterilizing mature seeds of soybean［J］.Plant Physiology Communications,2002,38(3):260-261.)
[6]Rech E L,Vianna G R,Aragao F J.High-efficiency transformation by biolistics of soybean,common bean and cotton transgenic plants［J］.Nature Protocols,2008,3(3):410-418.
[7]王关林,方宏筠.植物基因工程［M］.北京:科学出版社，2009:438-440.(Wang G L,Fang H J.Plant gene engineering［M］.Beijing:Science Press,2009:438-440.)
[8]Jefferson R A,Kavanagh T A,Bevan M W.GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants［J］.Embo Journal,1987,6(13):3901-3907.
[9]Murray M G,Tjompson W F.Rapid isolation of high molecular weight plant DNA［J］.Nucleic Acids Research,1980,19(8):4321-4325.
[10]Sambrook J,Russell David W.Molecular cloning:a laboratory manual.Vol.3.1989［K］.US Patents:Cold Spring Harbor Laboratory Press,2009.
［11］Hwang Y,Dawson J,Sigareva M,et al.Transformation of immature soybean seeds through organogenesis:US Patents，US 2008/0229447 A1［P］.2008-9-18.
［12］Liu H K,Yang C,Wei Z M.Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system［J］.Planta，2004，219(6):1042-1049.
［13］Ko T S,Lee S,Krasnyanski S,et al.Two critical factors are requiredfor efficient transformation of multiple soybean cultivars:Agrobacterium strain and orientation of immature cotyledonary explants ［J］.Theoretical and Applied Genetics，2003，107(3):439-447.
［14］Wang G L,Xu Y N.Hypocotyl-based Agrobacterium-mediated transformation of soybean(Glycine max)and application for RNA interference［J］.Plant Cell Reports，2008，27(7):1177-1184.
［15］Zhong H,Que Q D.Method for transforming soybean(Glycine max):US Patents，US 2009/0023212 A1［P］.2009-1-22.
［16］张敏,杨素欣,邵群,等.一种安全高效大豆转化筛选体系的建立［J］.生物技术通报,2012(5):66-70.(Zhang M,Yang S X,Shao Q,et al.A High-efficient and safe selection system of soybean transformation［J］.Biotechnology Bulletin,2012(5):66-70.)
［17］Shan Z,Raemakers K,Tzitzikas E N,et al.Development of a highly efficient,repetitive system of organogenesis in soybean［Glycine max(L.)Merr］［J］.Plant Cell Report,2005,24(9):507-512.
［18］叶美,张敏,杨素欣,等.大豆成熟种子胚尖基因枪法转化体系的优化［J］.大豆科学,2011,30（1）：20-23.(Ye M,Zhang M,Yang S X,et al.Optimization of biolistics transformation of embryonic tips of soybean[Glycine max（L.）Merrill]mature seeds［J］.Soybean Science，2011,30（1）：20-23.）
［19］郝荣华,邵群,杨素欣,等.根癌农杆菌介导的大豆子叶节转化体系的优化［J］.大豆科学,2012,31（2）：167-172.(Hao R H,Shao Q,Yang S X,et al.Optimization of Agrobacterium-mediated soybean transformation using the cotyledonary node［J］.Soybean Science，2012,31（2）：167-172.)

相似文献/References:

[1]刘章雄,李卫东,孙石,等.1983～2010年北京大豆育成品种的亲本地理来源及其遗传贡献[J].大豆科学,2013,32(01):1.[doi:10.3969/j.issn.1000-9841.2013.01.002]
　LIU Zhang-xiong,LI Wei-dong,SUN Shi,et al.Geographical Sources of Germplasm and Their Nuclear Contribution to Soybean Cultivars Released during 1983 to 2010 in Beijing[J].Soybean Science,2013,32(06):1.[doi:10.3969/j.issn.1000-9841.2013.01.002]
[2]李彩云,余永亮,杨红旗,等.大豆脂质转运蛋白基因GmLTP3的特征分析[J].大豆科学,2013,32(01):8.[doi:10.3969/j.issn.1000-9841.2013.01.003]
　LI Cai-yun,YU Yong-liang,YANG Hong-qi,et al.Characteristics of a Lipid-transfer Protein Gene GmLTP3 in Glycine max[J].Soybean Science,2013,32(06):8.[doi:10.3969/j.issn.1000-9841.2013.01.003]
[3]王明霞,崔晓霞,薛晨晨,等.大豆耐盐基因GmHAL3a的克隆及RNAi载体的构建[J].大豆科学,2013,32(01):12.[doi:10.3969/j.issn.1000-9841.2013.01.004]
　WANG Ming-xia,CUI Xiao-xia,XUE Chen-chen,et al.Cloning of Halotolerance 3 Gene and Construction of Its RNAi Vector in Soybean (Glycine max)[J].Soybean Science,2013,32(06):12.[doi:10.3969/j.issn.1000-9841.2013.01.004]
[4]张春宝,李玉秋,彭宝,等.线粒体ISSR与SCAR标记鉴定大豆细胞质雄性不育系与保持系[J].大豆科学,2013,32(01):19.[doi:10.3969/j.issn.1000-9841.2013.01.005]
　ZHANG Chun-bao,LI Yu-qiu,PENG Bao,et al.Identification of Soybean Cytoplasmic Male Sterile Line and Maintainer Line with Mitochondrial ISSR and SCAR Markers[J].Soybean Science,2013,32(06):19.[doi:10.3969/j.issn.1000-9841.2013.01.005]
[5]卢清瑶,赵琳,李冬梅,等.RAV基因对拟南芥和大豆不定芽再生的影响[J].大豆科学,2013,32(01):23.[doi:10.3969/j.issn.1000-9841.2013.01.006]
　LU Qing-yao,ZHAO Lin,LI Dong-mei,et al.Effects of RAV gene on Shoot Regeneration of Arabidopsis and Soybean[J].Soybean Science,2013,32(06):23.[doi:10.3969/j.issn.1000-9841.2013.01.006]
[6]杜景红,刘丽君.大豆fad3c基因沉默载体的构建[J].大豆科学,2013,32(01):28.[doi:10.3969/j.issn.1000-9841.2013.01.007]
　DU Jing-hong,LIU Li-jun.Construction of fad3c Gene Silencing Vector in Soybean[J].Soybean Science,2013,32(06):28.[doi:10.3969/j.issn.1000-9841.2013.01.007]
[7]张力伟,樊颖伦,牛腾飞,等.大豆“冀黄13”突变体筛选及突变体库的建立[J].大豆科学,2013,32(01):33.[doi:10.3969/j.issn.1000-9841.2013.01.008]
　ZHANG Li-wei,FAN Ying-lun,NIU Teng-fei?,et al.Screening of Mutants and Construction of Mutant Population for Soybean Cultivar "Jihuang13”[J].Soybean Science,2013,32(06):33.[doi:10.3969/j.issn.1000-9841.2013.01.008]
[8]盖江南,张彬彬,吴瑶,等.大豆不定胚悬浮培养基因型筛选及基因枪遗传转化的研究[J].大豆科学,2013,32(01):38.[doi:10.3969/j.issn.1000-9841.2013.01.009]
　GAI Jiang-nan,ZHANG Bin-bin,WU Yao,et al.Screening of Soybean Genotypes Suitable for Suspension Culture with Adventitious Embryos and Genetic Transformation by Particle Bombardment[J].Soybean Science,2013,32(06):38.[doi:10.3969/j.issn.1000-9841.2013.01.009]
[9]王鹏飞,刘丽君,唐晓飞,等.适于体细胞胚发生的大豆基因型筛选[J].大豆科学,2013,32(01):43.[doi:10.3969/j.issn.1000-9841.2013.01.010]
　WANG Peng-fei,LIU Li-jun,TANG Xiao-fei,et al.Screening of Soybean Genotypes Suitable for Somatic Embryogenesis[J].Soybean Science,2013,32(06):43.[doi:10.3969/j.issn.1000-9841.2013.01.010]
[10]刘德兴,年海,杨存义,等.耐酸铝大豆品种资源的筛选与鉴定[J].大豆科学,2013,32(01):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
　LIU De-xing,NIAN Hai,YANG Cun-yi,et al.Screening and Identifying Soybean Germplasm Tolerant to Acid Aluminum[J].Soybean Science,2013,32(06):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
[11]张忻爽,王萍,宋海星,等.卡那霉素和草铵膦对不同基因型大豆胚尖不定芽诱导的影响[J].大豆科学,2013,32(01):136.[doi:10.3969/j.issn.1000-9841.2013.01.032]
　ZHANG Xin-shuang,WANG Ping,SONG Hai-xing,et al.Effect of Knamycin and Glufosinate on Adventitious Buds Induction from Embryonic Tip of Different Genotype Soybean[J].Soybean Science,2013,32(06):136.[doi:10.3969/j.issn.1000-9841.2013.01.032]
[12]张兴政,王昌陵,卢福荣,等.根癌农杆菌介导大豆转化 LePT1 基因的研究[J].大豆科学,2014,33(01):31.[doi:10.11861/j.issn.1000-9841.2014.01.0031]
　ZHANG Xingzheng,WANG Changling,LU Furong,et al.Transformation of LePT1 Gene into Soybean via Agrobacterium mediation[J].Soybean Science,2014,33(06):31.[doi:10.11861/j.issn.1000-9841.2014.01.0031]
[13]钟 影,王 罡,季 静,等.草甘膦对不同基因型大豆不定芽再生的影响[J].大豆科学,2014,33(02):218.[doi:10.11861/j.issn.1000-9841.2014.02.0218]
　ZHONG Ying,WANG Gang,JI Jing,et al.Effect of Glyphosate on Adventitious Buds Induction from Embryonic Tip of Different Genotype Soybean[J].Soybean Science,2014,33(06):218.[doi:10.11861/j.issn.1000-9841.2014.02.0218]
[14]闫帆,孙昕,翟莹,等.大豆胚尖再生体系的研究[J].大豆科学,2011,30(05):757.[doi:10.11861/j.issn.1000-9841.2011.05.0757]
　YAN Fan,SUN Xin,ZHAI Ying,et al.Optimization on the Regeneration System of Soybean Embryonic Tips[J].Soybean Science,2011,30(06):757.[doi:10.11861/j.issn.1000-9841.2011.05.0757]
[15]郑丽红,季 静,王 罡,等.适于子叶节和胚尖再生体系的大豆基因型筛选[J].大豆科学,2012,31(02):212.[doi:10.3969/j.issn.1000-9841.2012.02.011]
　ZHENG Li-hong,JI Jing,WANG Gang,et al.Selection of Suitable Soybean Genotype Based on Cotyledon Node and Embryonic Tip Regeneration Systems[J].Soybean Science,2012,31(06):212.[doi:10.3969/j.issn.1000-9841.2012.02.011]
[16]王 伟,王 罡,季 静,等.大豆胚尖再生体系的优化及与子叶节再生体系的比较[J].大豆科学,2012,31(03):353.[doi:10.3969/j.issn.1000-9841.2012.03.004]
　WANG Wei,WANG Gang,JI Jing,et al.Optimization of Embryonic Tip Regeneration System and Comparison with Cotyledonary Node Regeneration System in Soybean[J].Soybean Science,2012,31(06):353.[doi:10.3969/j.issn.1000-9841.2012.03.004]
[17]姚丙晨,沈艳茹,韩 雪,等.大豆子叶节和胚尖再生体系的比较及大豆SR1基因的遗传转化[J].大豆科学,2012,31(03):364.[doi:10.3969/j.issn.1000-9841.2012.03.006]
　YAO Bing-chen,SHEN Yan-ru,HAN Xue,et al.Comparison with Cotyledonary Node and Embryonic Tip Regeneration System in Soybean\[Glycine max(L.)Merrill\] and Genetic Transformation of SR1 [J].Soybean Science,2012,31(06):364.[doi:10.3969/j.issn.1000-9841.2012.03.006]
[18]许 诺,张 君,王丕武.6-BA和IBA浓度对不同基因型大豆胚尖诱导丛生芽的影响[J].大豆科学,2012,31(04):678.[doi:10.3969/j.issn.1000-9841.2012.04.033]
　XU Nuo,ZHANG Jun,WANG Pi-wu.Effect of 6-BA and IBA Concentration on Shoots Induction from Embryonic Tips of Four Soybean Genotypes[J].Soybean Science,2012,31(06):678.[doi:10.3969/j.issn.1000-9841.2012.04.033]
[19]闫 帆,孙 昕,翟 莹,等.6-BA浓度及基因型对大豆胚尖诱导丛生芽的影响[J].大豆科学,2011,30(01):29.[doi:10.11861/j.issn.1000-9841.2011.01.0029]
　YAN Fan,SUN Xin,ZHAI Ying,et al.Effect of Different 6-BA Concentration and Genotypes on Shoots Induced from Embryonic Tips[J].Soybean Science,2011,30(06):29.[doi:10.11861/j.issn.1000-9841.2011.01.0029]
[20]朱红林,沙爱华,符秀梅,等.转录调控基因GmLEC1转化大豆及转化方法的比较[J].大豆科学,2010,29(01):7.[doi:10.11861/j.issn.1000-9841.2010.01.0007]
　ZHU Hong-lin,SHA Ai-hua,FU Xiu-mei,et al.Cloning and Transformation Study of Transcription Factor GmLEC1 in Soybean[J].Soybean Science,2010,29(06):7.[doi:10.11861/j.issn.1000-9841.2010.01.0007]

备注/Memo

基金项目：国家转基因重大专项（2014ZX0800943B）；国家自然科学基金（31271743）。

第一作者简介：孙式静（1987-），女，硕士，主要从事大豆遗传转化和分子育种研究。E-mail：liusunshijing@sina.cn。

更新日期/Last Update: 2014-12-25