农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究 (英文)

doi:10.17521/cjpe.2005.0044

植物生态学报 ›› 2005, Vol. 29 ›› Issue (2): 338-344.DOI: 10.17521/cjpe.2005.0044

• 论文 • 上一篇

农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究 (英文)

黄其满¹, 刘伟华², 孙辉¹, 邓新², 苏金¹^,^*()

1 中国农业科学院生物技术研究所,北京100081
2 中国农业科学院作物品种资源研究所,北京100081

收稿日期:2004-01-10 接受日期:2004-09-21 出版日期:2005-01-10 发布日期:2005-03-10
通讯作者: 苏金

AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSGENIC WHEAT PLANTS WITH GLUTAMINE SYNTHETASES CONFER TOLERANCE TO HERBICIDE

HUANG Qi-Man¹, LIU Wei-Hua², SUN Hui¹, DENG Xin², SU Jin¹^,^*()

1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2 Institule of Crop Germplasm Resources, Chinese Academy of Agriculural Sciences, Bejing 100081, China

Received:2004-01-10 Accepted:2004-09-21 Online:2005-01-10 Published:2005-03-10
Contact: SU Jin
About author:*E-mail:huangqiman@hotmail.com
Supported by:
National Special Program for Research and Industrialization of Transgenic Plants(J2000-B-006);National Natural Science Foundation of China(39980025)

摘要/Abstract

摘要：

谷氨酰胺合成酶 (Glutaminesynthetase, GS, E.C.6.3.1.2 ) 是植物氨同化过程中的关键酶, 对植物的氮素吸收和代谢起着至关重要的作用。谷氨酰胺合成酶还是除草剂草胺膦 (Phosphinothricin (PPT) 或Basta) 的靶标酶。前期工作已从我国特有的豌豆 (Pisumsatium) 品种中克隆了细胞质型谷氨酰胺合成酶 (GS1) cDNA和叶绿体型谷氨酰胺合成酶 (GS2 ) cDNA。为了验证谷氨酰胺合成酶的功能, 构建了同时含有GS1cDNA和GS2cDNA的植物表达载体p2GS。以该表达载体通过农杆菌介导法, 转化小麦 (Triticumaestivum) 的未成熟胚愈伤组织, 经PPT筛选及分化再生培养, 获得了抗PPT的转基因小麦植株 4 1株。PCR和基因组Southern杂交分析证实了GS1和GS2基因已经整合到转基因小麦的基因组。用除草剂草胺膦Basta溶液涂抹转p2GS小麦叶片, 结果证明GS转基因植株可以抗高达 0.3%的Basta溶液, 而对照植株叶片逐渐变黄直至枯死。转基因小麦植株能正常结实。上述实验结果表明 :1) GS基因在小麦植株中获得了有效表达, 从而赋予小麦植株抗PPT特性 ;2 ) GS基因能够作为研究小麦遗传转化的筛选标记基因。

关键词: 谷氨酰胺合成酶, 农杆菌介导的遗传转化, 转基因小麦, 除草剂抗性

Abstract:

Cloning of cytosolic (GS1) cDNA and chloroplast glutamine synthetase (GS2) cDNA from Pisum satium was carried out previously in our laboratory. To identify the functions of the GS genes, we first constructed a plant expression vector, p2GS, harboring two different isoenzymes, GS1 and GS2 cDNAs, under the control of two constitutive promoters of rice, Actin1 (Act1) and maize Ubiquitin (Ubi) genes. Then, using an Agrobacterium tumefaciens-mediated transformation method, we introduced GS1 and GS2 genes into wheat (Triticum aestivum) plants, producing 2GS-transgenic wheat plants using immature embryos as the explants. Presence of the transgenes GS1 and GS2 in wheat plants was confirmed by PCR and Southern blot hybridization analyses. Forty-one independent transgenic wheat plants with tolerance to an herbicide (Phosphinothricin, PPT) were generated. Results from Basta tests showed that the 2GS-transgenic wheat plants were endowed with herbicide-tolerant properties. Almost all of the transgenic plants were normal in morphology, and seed production was similar to that of the control wheat plants. Our study suggest that PPT resistance is conferred by effective expression of glutamine synthetases in transformed wheat plants, and glutamine synthetase genes can serve as a selective marker gene of wheat transformation system in our study.

Key words: Glutamine synthetases, Agrobacterium tumefaciens-mediated transformation, Transgenic wheat, Herbicide-tolerance

黄其满, 刘伟华, 孙辉, 邓新, 苏金. 农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究 (英文). 植物生态学报, 2005, 29(2): 338-344. DOI: 10.17521/cjpe.2005.0044

HUANG Qi-Man, LIU Wei-Hua, SUN Hui, DENG Xin, SU Jin. AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSGENIC WHEAT PLANTS WITH GLUTAMINE SYNTHETASES CONFER TOLERANCE TO HERBICIDE. Chinese Journal of Plant Ecology, 2005, 29(2): 338-344. DOI: 10.17521/cjpe.2005.0044

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图/表 3

Fig.1 Construction of p2GS expression vector

Fig.2 2-1 Comparision of wheat calli in differential medium with PPT CK (Upper) : the growth and development of control calli were inhibited in MS medium with PPT; Lower left, right: transgenic calli and shoots showing PPT resistance Fig.2-2 Regenerated transgenic wheat plantlets in regeneration medium with PPT Fig.2-3 Basta test: using 0.3% Basta solution scribble leaves of wheat plants. After 3 days the color of the leaves of untransformed control wheat plants (1-3) turned yellow and wither while the color of leaves (4-10) of the transgenic wheat plants showed Basta resistance, three of them (8-10) showed strong Basta resistance Fig.2-4 PCR analysis: 1-4. 2GS-transgenics DNA showing 1.1 kb for GS1, 1.3 kb for GS2 fragments; 5. non-transgenic plant DNA (Negative control) ; 6. p2GS (As a positive control) ; M. 1 kb maker Fig.2-5 Southern blot hybridization of transgenic wheat plants. Digestion of genomic DNA transformed wheat plants (Lane 1-9) or no-transgenic wheat plants (CK as negative control) with Hind III. Lane 1, 2, 5, 8, 9, 10 (p2GS as Positive control) gave positive signal while CK and Lane 3, 4, 6, 7 did not show any signal in Southern blot hybridization Fig.2-6 Regenerated transgenic wheat plants in soil

Table 1 Wheat calli selection and regeneration

Wheat cultivars	Number of transformed calli	Number of calli exhibiting PPT-resistance	Percentage of PPT- resistant calli	Number of PPT-resistant calli exhibiting shoot regeneration	Percentage of regenerated plantlets with PPT-resistance
96G25	430	83	19.30	20	24.10
Bobwhite	390	70	17.95	21	30.00
Total^*	820	153	18.63^*	41	27.05^*

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农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究 (英文)

AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSGENIC WHEAT PLANTS WITH GLUTAMINE SYNTHETASES CONFER TOLERANCE TO HERBICIDE

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