[1]段明.谷子EPSPS基因的分离、修饰及表达载体的构建[J].江苏农业科学,2016,44(05):51-55.
　Duan Ming.Separation， modification and construction of expression vector for millet EPSPS gene[J].Jiangsu Agricultural Sciences,2016,44(05):51-55.
点击复制

谷子EPSPS基因的分离、修饰及表达载体的构建()

《江苏农业科学》[ISSN:1002-1302/CN:32-1214/S]

卷:
第44卷
期数:
2016年05期
页码:
51-55
栏目:
生物技术
出版日期:
2016-05-25

文章信息/Info

Title:
Separation， modification and construction of expression vector for millet EPSPS gene
作者:
段明
山西农业大学实验教学中心，山西太谷 030801
Author(s):
Duan Ming
关键词:
谷子; 草甘膦; EPSPS基因; 表达载体
Keywords:
-
分类号:
Q78
DOI:
-
文献标志码:
A
摘要:
5-烯醇式丙酮酸莽草酸-3-磷酸合酶(EPSPS)是植物莽草酸合成途径中的一个重要酶，与芳香族氨基酸及其次生代谢物的合成有关。利用RT-PCR方法，从谷子中分离到5-烯醇式丙酮酸莽草酸-3-磷酸合酶基因SiEPSPS，全长1 573 bp，开放阅读框1 536 bp，编码511个氨基酸。同源序列比较发现，SiEPSPS氨基酸序列与小麦、短柄草、高粱的EPSPS序列高度同源。结构同源性分析表明，SiEPSPS与其他植物一样，均具有2个序列保守的结构域。对其启动子序列分析显示，该片段富含ABRE、TC-rich repeats、TCA-element等响应胁迫的顺式作用元件。针对草甘膦结合位点，对SiEPSPS基因进行定点修饰。为研究其功能，构建植物表达载体pWM101-EPSPS，并导入农杆菌进行检测。测序结果比对表明，突变型EPSPS基因在173位的脯氨酸变为丝氨酸，为后续EPSPS的真核表达奠定了基础。
Abstract:
-

参考文献/References:

［1］Duke S O，Powles S B. Glyphosate：a once-in-a-century herbicide［J］. Pest Management Science，2008，64(4)：319-325.
［2］Brookes G，Barfoot P. The global income and production effects of genetically modified(GM) crops：1996—2011［J］. GM Crops & Food，2013，4(1)：74-83.
［3］Gardner J G，Nelson G C. Herbicides，glyphosate resistance and acute mammalian toxicity：simulating an environmental effect of glyphosate-resistant weeds in the USA［J］. Pest Management Science，2008，64(4)：470-478.
［4］Bonny S. Herbicide-tolerant transgenic soybean over 15 years of cultivation：pesticide use，weed resistance，and some economic issues［M］. Sustainability，2011，3(9)：1302-1322.
［5］Steinrücken H C，Amrhein N. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase［J］. Biochemical and Biophysical Research Communications，1980，94(4)：1207-1212.
［6］Alibhai M F，Stallings W C. Closing down on glyphosate inhibition：with a new structure for drug discovery［J］. Proceedings of the National Academy of Sciences of the United States of America，2001，98(6)：2944-2946.
［7］Howe A R，Gasser C S，Brown S M，et al. Glyphosate as a selective agent for the production of fertile transgenic maize(Zea mays L.) plants［J］. Molecular Breeding，2002，10(3)：153-164.
［8］Wang H Y，Li Y F，Xie L X，et al. Expression of a bacterial aroA mutant，aroA-M1，encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants［J］. Journal of Plant Research，2003，116(6)：455-460.
［9］Ye G N，Hajdukiewicz P T，Broyles D，et al. Plastid-expressed 5-enol-pyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco［J］. Plant Journal，2001，25(3)：261-270.
［10］Garbe T，Jones C，Charles I，et al. Cloning and characterization of the aroA gene from Mycobacterium tuberculosis［J］. Journal of Bacteriology，1990，172(12)：6774-6782.
［11］Ream J E，Steinrücken H C，Porter C A，et al. Purification and properties of 5-enolpyruvylshikimate-3-phosphate synthase from dark-grown seedlings of Sorghum bicolor［J］. Plant Physiology，1988，87(1)：232-238.
［12］Xu J W，Feng D J，Li X G，et al. Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene［J］. Science in China：Series C，Life Sciences，2002，45(3)：251-259.
［13］Gong Y，Liao Z，Chen M，et al. Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminate［J］. Biologia Plantarum，2006，50(4)：542-550.
［14］Funke T，Han H，Healy F M L，et al. Molecular basis for the herbicide resistance of Roundup Ready crops［J］. Proceedings of the National Academy of Sciences of the United States of America，2006，103(35)：13010-13015.
［15］Sun Y C，Chen Y C，Tian Z X，et al. Novel AroA with high tolerance to glyphosate，encoded by a gene of Pseudomonas putida 4G-1 isolated from an extremely polluted environment in China［J］. Applied and Environmental Microbiology，2005，71(8)：4771-4776.
［16］Chen J C，Huang H J，Zhang C X，et al. Mutations and amplification of EPSPS gene confer resistance to glyphosate in goosegrass (Eleusine indica)［J］. Planta，2015，242(4)：859-868.
［17］Castle L A，Siehl D L，Gorton R，et al. Discovery and directed evolution of a glyphosate tolerance gene［J］. Science，2004，304(5674)：1151-1154.
［18］Baerson S R，Rodriguez D J，Tran M，et al. Glyphosate-resistant goosegrass.Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase［J］. Plant Physiology，2002，129(3)：1265-1275.
［19］González T F，Gil-Humanes J，Barro F，et al. Target site mutation and reduced translocation are present in a glyphosate-resistant Lolium multiflorum Lam.biotype from Spain［J］. Plant Physiology and Biochemistry，2012，58(3)：16-22.
［20］陈建华，卢敏，蔡志斌. 维生素C拮抗草甘膦对蚯蚓急性毒性的作用［J］. 江苏农业科学，2015，43(7)：411-413，425.
［21］王莹，袁英. 抗除草剂转基因玉米的快速鉴定方法［J］. 江苏农业科学，2014，42(4)：54-56.
［22］Alarcon R R，Garcia A，Urzua J A. Resistance to glyphosate in junglerice(Echinochloa colona) from California［J］. Weed Science，2013，61(1)：48-54.
［23］Howe A R，Gasser C S，Brown S M，et al. Glyphosate as a selective agent for the production of fertile transgenic maize(Zea mays L.) plants［J］. Molecular Breeding，2002，10(3)：153-164.
［24］Kahrizi D，Salmanian A H，Afshari A，et al. Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate［J］. Plant Cell Reports，2007，26(1)：95-104.
［25］Yu Q，Jalaludin A，Han H，et al. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance［J］. Plant Physiology，2015，167(4)：1440-1447.

相似文献/References:

[1]李君,宋青春,陈秀敏.农药草甘膦对刺参成参的急性毒性[J].江苏农业科学,2013,41(04):231.
[2]李会芬,时丽冉,崔兴国,等.水分胁迫对不同品种谷子萌发期的影响[J].江苏农业科学,2013,41(05):67.
　Li Huifen,et al.Effect of water stress on germination stage of different millet cultivars[J].Jiangsu Agricultural Sciences,2013,41(05):67.
[3]王军华,王易芬,陈蕾蕾,等.除草剂草甘膦微生物降解技术研究进展[J].江苏农业科学,2016,44(04):8.
　Wang Junhua,et al.Research progress on microbial degradation of glyphosate[J].Jiangsu Agricultural Sciences,2016,44(05):8.
[4]赵敏,李书田,王显瑞,等.谷子新品种峰红谷的选育及栽培要点[J].江苏农业科学,2013,41(11):116.
　Zhao Min,et al.Breeding and cultivation techniques of new millet cultivar “Fenghonggu”[J].Jiangsu Agricultural Sciences,2013,41(05):116.
[5]张艾英,郭素芬,张莉,等.膜侧沟播对旱地春谷生理生态特性及产量的影响[J].江苏农业科学,2016,44(06):148.
　Zhang Aiying,et al.Effects of ridge film mulching and furrow seeding on eco-physiological property and yield of spring foxtail millet in dry land[J].Jiangsu Agricultural Sciences,2016,44(05):148.
[6]呼红梅,王莉.氮、磷、钾对盐胁迫谷子幼苗形态和生理指标的影响[J].江苏农业科学,2016,44(02):117.
　Hu Hongmei,et al.Effects of nitrogen，phosphorus and potassium on seedling morphology and physiology indices of millet under salt stress[J].Jiangsu Agricultural Sciences,2016,44(05):117.
[7]王莹,袁英.抗除草剂转基因玉米的快速鉴定方法[J].江苏农业科学,2014,42(04):54.
　Wang Ying,et al.Rapid identification method of transgenic maizes with herbicide resistance[J].Jiangsu Agricultural Sciences,2014,42(05):54.
[8]王彦杰,毕少杰,洪秀杰,等.不同浓度沼液浸种和喷施对谷子生长的影响[J].江苏农业科学,2015,43(10):119.
　Wang Yanjie,et al.Effect of soaking and spraying with different concentrations of biogas slurry on growth of millet[J].Jiangsu Agricultural Sciences,2015,43(05):119.
[9]陈建华,卢 敏,蔡志斌.维生素C拮抗草甘膦对蚯蚓急性毒性的作用[J].江苏农业科学,2015,43(07):411.
　Chen Jianhua,et al.Effect of vitamin C antagonist acute toxicity of glyphosate to Eisenia fetida[J].Jiangsu Agricultural Sciences,2015,43(05):411.
[10]刘志军,黄艳芳,刘金红.草甘膦催化工艺优化及贵金属催化剂再生研究[J].江苏农业科学,2014,42(07):146.
　Liu Zhijun,et al.Study on optimization of glyphosate catalytic process and regeneration of noble metal catalyst[J].Jiangsu Agricultural Sciences,2014,42(05):146.

备注/Memo

备注/Memo:
收稿日期：2016-02-17
基金项目：山西省青年科技研究基金(编号：2015021142)；山西农业大学科技创新基金(编号：20142-12)。
作者简介：段明(1984—)，男，山东威海人，博士，讲师，主要从事植物抗逆分子生物学研究。E-mail：duanming840305@163.com。
更新日期/Last Update: 2016-05-25