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| 水稻几丁质酶的原核表达、复性及体外抑菌活性的研究 | |
| 引用本文： | 刘宝业,梁国鲁,张增艳.水稻几丁质酶的原核表达、复性及体外抑菌活性的研究[J].核农学报,2009,23(3):369-374. |
| 作者姓名： | 刘宝业 梁国鲁 张增艳 |
| 作者单位： | 1. 中国农业科学院作物科学研究所,农作物基因资源与基因改良国家重大科学工程,北京,100081;西南大学园艺园林学院,重庆,400716 2. 西南大学园艺园林学院,重庆,400716 3. 中国农业科学院作物科学研究所,农作物基因资源与基因改良国家重大科学工程,北京,100081 |
| 基金项目： | 国家转基因生物重大专项课题，重点课题 |
| 摘 要： | 利用RT-PCR反应，从纹枯菌诱导的水稻叶片cDNA中克隆了1个水稻几丁质酶基因RC7的全长编码序列（ORF）。通过亚克隆方法，RC7与原核表达载体pGEX-4T-1上的GST融合，构建成GST-RC7重组蛋白的原核表达载体pGST-RC7，然后，将其转化到大肠杆菌细胞中。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，获得了以包涵体形式高效表达的GST-RC7。经过洗涤、溶解（变性）、复性及纯化等处理，包涵体中GST-RC7得到溶解、复性和纯化。纯化的复性GST-RC7蛋白具有几丁质酶活性。对6种重要作物病原真菌进行体外抑菌活性分析的结果表明， RC7可显著抑制水稻纹枯病菌、小麦纹枯菌、小麦赤霉菌、棉花黄萎菌、烟草赤星菌的菌丝生长，说明RC7可作为上述植物病害抗性基因育种的重要基因。 |
| 关 键 词： | 水稻几丁质酶基因 表达 蛋白质纯化 复性 体外抑制真菌活性 |
| 收稿时间： | 2008-10-14 |
Prokaryotic Expression, Refolding and Antifungal Activity of a Rice Chitinase in vitro |
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| LIU Bao-ye,Liang Guo-lu,ZHANG Zeng-yan.Prokaryotic Expression, Refolding and Antifungal Activity of a Rice Chitinase in vitro[J].Acta Agriculturae Nucleatae Sinica,2009,23(3):369-374. | |
| Authors: | LIU Bao-ye Liang Guo-lu ZHANG Zeng-yan |
| Institution: | 1;2;1;1.National Key Science Facility of Crop Gene Resources and Gene Improvement;Institute of Crop Science;Chinese Academy of Agricultural Sciences;Beijing 100081;2.College of Horticulture and Landscape Architecture;Southwest University;Chongqing 400716 |
| Abstract: | The full open-reading frame (ORF) sequence of a rice chitinase gene RC7 was isolated from the rice leaf cDNA induced by Rhizoctonia solani through subclone method. The RC7 was fused in frame to the ORF of Glutathione S-transferase （GST）in the prokaryotic expression vector pGEX-4T-1, in which the new recombinant vector pGST-RC7, containing the recombinant protein GST-RC7, was constructed. Then the pGST-RC7 vector was transformed into cells of Escherichia coli. After induced by isopropyl-β-D-thiogalactoside (IPTG), the recombinant protein GST-RC7 was highly expressed in the form of inclusion bodies. The protein GST-RC7 in the inclusion bodies could be solubilized, renatured and purified by a serial of treatments, including washing, denaturing and renaturing as well as purification. The renaturing protein possessed chitinase activity. The results of antifungal assay in vitro showed that the chitinase RC7 exerted significant inhibition to the mycelia growth of Rhizoctonia solani (rice sheath blight), Rhizoctonia cerealis (wheat sharp eyespot), Verticillium dahliae (cotton wilt), Fusarium graminearum (wheat scab) and Alternaria longipes (tobacco brown spot). RC7 may be useful for enhancing fungal resistance in several crop species. |
| Keywords: | rice chitinase gene expression protein purification renaturation antifungal activity in vitro |
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