[1]薛仁镐 　谢宏峰.利用改良的草丁膦筛选系统快速而有效筛选转基因大豆[J].大豆科学,2006,25(04):373-378.[doi:10.11861/j.issn.1000-9841.2006.04.0373]
　Xue Rengao Xie Hongfeng.Rapid And Efficient Selection For TransgenicSoybean Plants With The ImprovedGlufosinate Selection System*[J].Soybean Science,2006,25(04):373-378.[doi:10.11861/j.issn.1000-9841.2006.04.0373]
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利用改良的草丁膦筛选系统快速而有效筛选转基因大豆

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第25卷 期数: 2006年04期 页码: 373-378 栏目: 出版日期: 2006-11-25
Title:
Rapid And Efficient Selection For TransgenicSoybean Plants With The ImprovedGlufosinate Selection System*
作者:
薛仁镐 　谢宏峰
(莱阳农学院生命科学院, 青岛 266109)
Author(s):
Xue Rengao 　Xie Hongfeng
(Laiyang Agricultural College , Qingdao 266109)
关键词:
农杆菌 ; 子叶节; 草丁膦; 大豆; 遗传转化
Keywords:
Agrobacterium tumef aciens ; Cotyledonary node; Glufosinate; Soybean; Transfor-mation
DOI:
10.11861/j.issn.1000-9841.2006.04.0373
文献标志码:
A
摘要:
　为提高大豆(Glycine max (L.) Merrill)遗传转化效率建立了一种快速而有效的草丁膦筛选系统。将刺伤的子叶节外植体接种于含有 pCAMBIA3201 载体的 LBA4404 农杆菌溶液。目前利用草丁膦筛选转基因大豆的常规方法是在含有5 mg /L 草丁膦的芽诱导培养基和含有 3～ 5 mg /L 草丁膦的芽伸长培养基上进行筛选 。利用此常规筛选方法所获得的大多数植株是非转化体, 从而导致转化效率变低, 为 1. 6%;而且这种方法极大地延迟了芽的伸长过程, 使多数不定芽的伸长发生在农杆菌处理后的 21～ 41 周 。为此 ,对常规草丁膦筛选方法进行了改良 。首先将外植体放置在不含有除草剂的芽诱导培养基上培养 3 周, 然后转到含有 4 mg /L 草丁膦的芽伸长培养基上进行筛选 。此时,多数不定芽仅在 7～ 12 周内便可伸长 。当芽长到3～ 5 cm 时 ,从外植体上切下来转到含有 1 ～ 5 mg /L 草丁膦的根诱导培养上进行进一步的筛选。结果表明,在添加有 3 mg /L 草丁膦根培养基上,转化效率达到最大值为6. 7%。不定芽对根培养基中的除草剂反应迅速,仅在 10d 天内所有非转化体都变枯死亡 。利用这种改良的筛选系统, 大多数转基因植株仅在 8～ 16 周内便可获得 。Southern 杂交结果证实了外源基因稳定地整合在大豆基因组中 。GUS 检测和除草剂抗性分析结果表明 ,被整合的外源基因在大豆细胞中得到了稳定的表达。
Abstract:
　A rapid and efficient glufosinate selection system for obtaining high frequency of trans-formants insoybean [ Glycine max (L. ) Merrill] was developed. The cotyledonary node cellswere wounded and inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a bina-ry vector pCAMBIA3201 that contained a selectable bar gene and a gus reporter gene. The pres-ent standard selection based on glufosinate was performed at 5 mg /L glufosinate during shoot in-duction and at 3～ 5 mg /L glufosinate during shoot elongation. Many non-transformants were es-caped with this standard selection system which resulted in low transformation efficiency of1. 6%, and a delayed shoot elongation with majority of transformed shoots elongation occurredfrom 21 to 41 weeks after Agrobacterium inoculation , and thus an improved glufosinate selectionsystem was provided in this paper. After 3 weeks on shoot induction medium without glufosi-nate , the explants were transferred to shoot elongation medium containing 4 mg /L glufosinate forthe first selection. The elongation of majority of transformed shoots occurred only from 7 to 12weeks without glufosinate selection during shoot induction. The transgenic shoots were effective-ly screened by placing the excised shoots on the root induction medium (RIM containing 3 mg /Lglufosinate to facilitate direct uptake of the selective agent that resulted in the high transforma-tion efficiency of 6. 7%. Shoots on the RIM rapidly responded to the selective agent applied , allthe glufosinate-sensitive shoots were completely necrotic within 10 days after selection. The ma-jority of transgenic plantlets were obtained only 8 ～ 16 weeks under the improved selection sys-tem. Genomic Southern blot analysis confirmed stable integration of the transgenes in the genomeof soybean. Stable expression was confirmed by GUS expression and herbicide application.

参考文献/References:

1　 Olhoft PM , Flagel LE, Donovan CM , et al. Efficient soybeantransformation using hyg romy cin B selection in the coty ledonary-node method[ J] . Planta , 2003, 216:723- 735.

2　 Hinchee MAW, Connor-Ward DV , Newell CA , et al. Produc-tion of transgenic soybean plants using Agrobacterium-mediatedDNA transfer[ J] . Bio /Technology , 1988, 6:915- 922.
3　 Di R, Purcell V , Collins GB. Production of transgenic soybeanlines expressing the bean pod mottle virus coat protein precursorgene[ J] . Plant Cell Rep, 1996, 15:746- 750.
4　 Liu HK , Yang C, Wei ZM. Efficient Agrobacterium tumef a-ciens-mediated transfo rmation of soybeans using an embryonic tipregeneration sy stem[ J] . Planta, 2004, 219:1042 - 1049.
5　 Clemente TE, LaVallee BJ, Howe AR, et al. Progeny analysisof glyphosate selected transgenic soybeans derived fromAgrobacterium-mediated transformation[ J] . Crop Sci. , 2000,40:797- 803.
6　 Finer JJ, M cMullen MD. Transformation of soybean via particlebombardment of embryogenic suspension culture tissue[ J] . InVitro Cell Dev Biol, 1991, 27P :175 - 182.
7　 T rick HN , Finer JJ. Sonication-assisted Agrobacterium-media-ted transformation of soybean [ (Gly cine max (L.) Merrill) em-bryogenic suspension culture tissue[ J] . Plant Cell Rep, 1998,17:482- 488.
8　 Zhang Z , Xing A, Staswick PE, et al. The use of glufosinate asa selective agent in Agrobacterium-mediated transformation ofsoybean[ J] . Plant Cell Tissue Organ Cult, 1999, 56:37 - 46.
9　 Olhoft PM , Somers DA. L-Cy steine increases Agrobacterium-mediated T - DNA delivery into soybean coty ledonary-node cells[ J] . Plant Cell Rep, 2001, 20:706- 711.
10 Zeng P , Vadnais DA , Zhang Z , et al. Refined glufosinate selec-tion in Agrobacterium-mediated transformation of soy bean[ Gly-cine max (L.) Merrill] [ J] . Plant Cell Rep, 2004, 22:478 -482.
11 Paz MM , Shou H, Guo Z , et al. Assessment of conditions af -fecting Agrobacterium-mediated soybean transformation usingthe cotyledonary node explant[ J] . Euphytica , 2004, 136:167-179.
12 Olhoft PM , Lin K , Galbraith J, et al. The role of thiol com-pounds increasing Agrobacterium-mediated transformation ofsoybean cotyledonary-node cells[ J] . Plant Cell Rep, 2001, 20 731- 737.
13 M urashige T , Skoog F. A revised medium for rapid grow th andbioassay s with tobacco tissue cultures[ J] . Plant Physiol, 1962,15:473 - 497.
14 Gamborg , OL , Miller RA , Ojima. RA. Nutrient requirementsof suspension culture of soybean root cells[ J] . Exp Cell Res,1968, 50:151- 158.
15 Jefferson RA , Kavanagh T A , Bevan MW. GUS fusion:β-glucu-ronidase as a sensitive and versatile gene fusion marker in higherplants[ J] . EM BO J, 1987, 6:3901- 3907.
16 Sambrook J, Russell DW. Molecular Cloning :a laboratory man-ual. 3rd ed[ R] . NY :Cold Spring Harbor Lab Press, 2001.
17 Chen SY. Hig h-efficiency Agrobacterium-mediated transforma-tion of soybean[ J] . Acta Botanica Sinica, 2004, 46(5):610 -617.
18 Shelp BJ, Swanton CJ, Hall JC. Glufosinate (Phosphinothricin)mobility in young soy bean shoots[ J] . J Plant Phy siol, 1992,139:626- 628.

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备注/Memo

基金项目:山东省教育厅基金(J05K04)和莱阳农学院基金(630308)

作者简介:薛仁镐,(1965 -), 男, 博士, 副教授, 主要从事大豆基因工程。E - mail:xuerengao @163. com

更新日期/Last Update: 2016-12-16