[1]张宪丽,郭勇,金龙国,等.未成熟种子子叶节转化体系优化及转EPSPS/GAT基因大豆新材料创制[J].大豆科学,2015,34(02):199-204.[doi:10.11861/j.issn.1000-9841.2015.02.0199]
　ZHANG Xian-li,GUO Yong,JIN Long-guo,et al.Optimization of Immature Seed Cotyledon-node Transformation System and Development of Genetically Modified Soybean by Transformation of EPSPS and GAT genes[J].Soybean Science,2015,34(02):199-204.[doi:10.11861/j.issn.1000-9841.2015.02.0199]
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未成熟种子子叶节转化体系优化及转EPSPS/GAT基因大豆新材料创制

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第34卷 期数: 2015年02期 页码: 199-204 栏目: 出版日期: 2015-04-25
Title:
Optimization of Immature Seed Cotyledon-node Transformation System and Development of Genetically Modified Soybean by Transformation of EPSPS and GAT genes
作者:
张宪丽1; 2; 郭勇2; 金龙国2; 张丽娟2; 邱丽娟1; 2
1.东北农业大学 农学院，黑龙江 哈尔滨 150030；?
2.中国农业科学院 作物科学研究所/国家农作物基因资源与遗传改良重大科学工程/农业部北京大豆生物学重点实验室，北京 100081
Author(s):
ZHANG Xian-li1; 2; GUO Yong2; JIN Long-guo2; ZHANG Li-juan2; QIU Li-juan2
1College of Agriculture, Northeast Agricultural University, Harbin 150030, China;?
2The National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI) / MOA Key Lab of Soybean Biology (Beijing), Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
关键词:
大豆; 未成熟种子; 子叶节; 遗传转化
Keywords:
Soybean;  Immature seed;  Cotyledonary node;  Transformation
分类号:
S565.1
DOI:
10.11861/j.issn.1000-9841.2015.02.0199
文献标志码:
A
摘要:
以丛生芽率为指标，将农杆菌介导的大豆未成熟种子子叶节转化与成熟种子转化体系进行比较，明确了诱导过程中草甘膦筛选浓度、未成熟种子生理状态，改进大豆外植体的获得方式并减少了组培环节，建立了未成熟种子子叶节转化体系。利用Jack黄熟后期的种子作为受体材料，在15 mg?L-1的草甘膦筛选浓度下获得了9株PCR检测阳性植株并且生长正常可育，T0代经测序及Southern Blot分析，进一步证明外源基因已整合进大豆基因组中，其中2个株系的T1代植株经PCR检测符合3∶1的分离比，且表型鉴定筛选出抗性转基因植株，为抗草甘膦转基因大豆育种提供了新材料。
Abstract:
Simple and efficient genetic transformation systems are very important for breeding transgenic soybeans and performing functional genomics study. In order to develop a concise and highly efficient Agrobacterium-mediated transformation system in soybean, transformation methods using immature seeds and mature seeds were compared.The effect of different factors on the .Agrobacterium-mediated transformation of immature seeds was investigated using the rate of resistant bud formation. These factors affecting transformation include selection pressure and the physiological state of the immature seeds Finally, the Agrobacterium-mediated transformation system of immature seed was optimized. Nine transgenic plants were screened with 15 mg?L-1?glyphosate and they grew normally and fertilityPCRsequencing and Southern Blot analysis also showed that the foreign genes had been integrated into the genome of the T0?individuals PCR analysis of 2 lines from the T1?generation also showed that the separation ratio fit 3∶1 The glyphosate tolerant soybeans obtained in this study by phenotype identification provide new materials for breeding of transgenic soybean with glyphosate tolerance.

参考文献/References:

［1］Sairam R V, Franklin G, Hassel R, et al.A study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration –via-organogenesis from soybean cotyledonary nodal callus ［J］ Plant Cell, Tissue and Organ Culture, 2003, 75(1): 79-85.

［2］Wang G, Xu Y.Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max L Merrill) and application for RNA interference ［J］ Plant Cell Reports, 2008, 27(7): 1177-1184.
［3］Cheng M, Lowe B A, Spencer T M, et al. Factors influencing Agrobacterium-mediated transformation of monocotyledonous species ［J］In Vitro Cellular & Developmental Biology-Plant， 2004, 40(1): 31-45.
［4］Donaldson P A, Simmonds D H.Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean ［J］ Plant Cell Reports， 2000, 19(5): 478-484.
［5］Ke J, Khan R, Johnson T, et al. High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens［J］ Plant Cell Reports， 2001, 20(2): 150-156.
［6］Olhoft P M, Flagel L E, Donovan C M, et al.Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method ［J］ Planta， 2003, 216(5): 723-735.
［7］Xue R, Xie H, Zhang B.A multi-needle-assisted transformation of soybean cotyledonary node cells ［J］Biotechnology Letters， 2006, 28(19): 1551-1557.
［8］Yamada T, Watanabe S, Arai M, et al. Cotyledonary node pre-wounding with a micro-brush increased frequency of Agrobacterium-mediated transformation in soybean ［J］Plant Biotechnology， 2010, 27: 217-220.
［9］Ko T, Lee S, Krasnyanski S, et al.Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium-strain and orientation of immature cotyledonary explant ［J］Theoretical and Applied Genetics， 2003, 107(3): 439-447.
［10］Yan B, Reddy M S, Collins G B, et al.Agrobacterium tumefaciens-mediated transformation of soybean ［Glycine max(L) Merrill］ using immature zygotic cotyledon explants ［J］Plant Cell Reports， 2000, 19(11): 1090-1097.
［11］Mariashibu T S, Subramanyam K, Arun M, et al.Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars ［J］Acta Physiologiae Plantarum， 2013, 35(1): 41-54.
［12］Franklin G, Carpenter L, Davis E, et al. Factors influencing regeneration of soybean from mature and immature cotyledons ［J］ Plant Growth Regulation， 2004, 43(1): 73-79.
［13］王卢平，乔亚科，李桂兰，等.大豆未成熟种子子叶节不定芽再生的初步研究［J］ 大豆科学， 2011，30(3): 369-373（Wang L P, Qiao Y K, Li G L, et al.Shoots regeneration of cotyledon node from soybean immature seed ［J］ Soybean Science， 2011, 30(3): 369-373）
［14］钟磊，王林红，乔亚科，等.农杆菌介导大豆未成熟子叶节的遗传转化［J］河北科技师范学院学报， 2013，27(1): 1-4（Zhong L, Wang L H, Qiao Y K, et al. Agrobacterium-mediated transformation of soybean in immature seed cotyledon node ［J］ Journal of Hebei Normal University of Science and Technology, 2013, 27(1): 1-4）
［15］胡晋. 种子生物学［M］北京：高等教育出版社， 2005（Hu J.Seed biology ［M］ Beijing: Higher Education Press， 2005）
［16］Paz M M, Shou H, Guo Z, et al. Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant ［J］ Euphytica， 2004, 136(2): 167-179
［17］Joyce P, Kuwahata M, Turner N, et al.Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane ［J］ Plant Cell Reports， 2010, 29(2): 173-183
［18］陆玲鸿，韩强，李林，等. 以草甘膦未筛选标记的大豆转基因体系的建立及抗除草剂转基因大豆的培育［J］ 中国科学: 生命科学， 2014，44(4): 406-415（Lu L H, Han Q, Li L, et al.Establishment of an efficient transformation protocol for soybean using glyphosate as selective agent and the development of glyphosate-tolerant transgenic soybean lines ［J］China Science, Life Science， 2014, 44(4): 406-415）
［19］马林，周练，周正剑，等. 抗除草剂转基因水稻和大豆快速准确检测技术研究［J］浙江大学学报 (农业与生命科学版)， 2012，38(6): 647-654（Ma L, Zhou L, Zhou Z J, et al.Establishment of methods for identifying rapidly and precisely transgenic rice and soybean containing herbicide-resistant gene Bar and EPSPS ［J］ Journal of Zhejiang University (Agriculture and Life Sciences), 2012, 38(6): 647-654）

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备注/Memo

基金项目：转基因生物新品种培育重大专项（2011ZX08004-001）。

第一作者简介：张宪丽（1987-），女，硕士，主要从事大豆转基因研究。E-mail：zhangxianli2007@163.com。
?通讯作者：邱丽娟（1963-），女，研究员，博导，主要从事大豆基因资源挖掘与利用工作。E-mail：qiulijuan@caas.cn。

更新日期/Last Update: 2015-06-07