| 摘要: |
| 【目的】构建aiiA基因的原核表达载体，并进行诱导表达，检测aiiA蛋白的抗病性，为进一步通过转基因技术培育转aiiA基因植株奠定基础。【方法】从pMDTM-19-T Vector＋aiiA质粒中酶切获得aiiA基因，将其与pGEX-4T-1表达载体连接构建重组原核表达载体pGEX-4T-1＋aiiA，经双酶切及测序鉴定后，进行诱导表达，并对表达产物的功能进行鉴定。【结果】双酶切与PCR检测结果表明，重组原核表达载体pGEX-4T-1＋aiiA构建成功。表达条件优化结果表明，在25 ℃下用0.2 mmol/L IPTG诱导9 h，aiiA蛋白的表达量最高。aiiA重组蛋白抑菌试验结果表明，其能够降解细菌的AHLs信号分子，猝灭细菌的群体感应，明显减弱病菌的致病力。转aiiA基因尾巨桉抗性检测结果表明，转基因植株抗性明显增强，表现为发病时间延迟，病情指数降低，评价为中等抗病水平。【结论】成功构建了aiiA基因原核表达载体，其诱导表达产物能猝灭细菌的群体感应，明显减弱病菌的致病力。 |
| 关键词: 群体感应 aiiA基因 N-酰基高丝氨酸内酯 N-酰基高丝氨酸内酯酶 |
| DOI： |
| 分类号: |
| 基金项目:国家“星火”计划项目（2012GA780019，2011GA780057）；广东高校边缘热带特色植物资源工程技术开发中心项目（GCZX-B1002）；广东省科技计划项目（2012A020602108）；广东省自然科学基金项目（S2013040014690，S2012010008737）；湛江市科技攻关项目（2011C3104010） |
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| Construction and expression of prokaryotic vector for aiiA gene |
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OUYANG Le-jun,LIANG Zuo-ling,HUANG Zhen-chi,et al
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| Abstract: |
| 【Objective】Construction expression of prokaryotic vector for aiiA gene,then the recombinant plasmid was induced to express disease resistance identification.This work laid the foundations for future transgenic research on aiiA gene by the plant transgenic technology.【Method】The study cloned the aiiA gene from pMDTM-19-T Vector＋aiiA by PCR,inserted into pGEX-4T-1 and constructed prokaryotic expression vector pGEX-4T-1＋aiiA.The recombinant plasmid was identified by restriction endonucleases analysis and protein expression was induced and the induce product was identified.【Result】Enzyme digestion and PCR showed that the recombinant plasmid pGEX-4T-1＋aiiA was constructed successfully.The maximum expression was obtained when induced by 0.2 mmol/L IPTG at 25 ℃ for 9 hours.Antibacterial test of recombinant protein showed that recombinant plasmid pGEX-4T-1＋aiiA could degrade AHLs by suppressing quorum sensing and attenuating pathogenicity of bacteria.Plant inoculation experiment showed that resistance to disease of transgenic Eucalyptus urophylla×E.grandis with aiiA was significantly enhanced by delaying the wilt symptom and decreasing disease index. 【Conclusion】The recombinant plasmid pGEX-4T-1＋aiiA was constructed correctly. |
| Key words: quorum sensing aiiA gene N-acyl-homoserine lactones N-acyl-homoserine enzymes |
