转基因抗虫棉根际卡那霉素抗性细菌种群动态及nptⅡ基因漂移检测

【目的】研究转基因抗虫棉根际卡那霉素抗性细菌的种群动态,检测转基因棉卡那霉素抗性细菌nptII基因漂移。【方法】采用常规培养方法分析了转基因抗虫棉GK12、GK19、33B、SGK321,常规棉泗棉3号、33、石远321等7个棉花品种根际卡那霉素抗性细菌种群在棉花整个生育期的种群动态变化。以质粒pBI121为对照,设计nptII基因特异性引物,采用PCR方法对分离的根际卡那霉素抗性细菌菌株进行nptII基因漂移检测。【结果】7个棉花品种根际卡那霉素抗性细菌数量随时间延长而减少,同一棉花品种不同采样时间卡那霉素抗性细菌数量差异显著,而同一采样时间抗虫棉和对应的受体棉间差异不显著。Simpson、Shannon-Wiener多样性指数以及Pielou均匀度指数计算结果表明,受体棉根际卡那霉素抗性细菌群落的多样性高于转基因棉。PCR检测结果表明,21株卡那霉素抗性细菌菌株中18株发现有阳性片段,但其测序结果与卡那霉素抗性基因序列比对的同源性未能达到100%,不能判断nptII是否发生了转移。【结论】根际卡那霉素抗性细菌的数量在转基因棉与对应受体棉之间差异不显著,受体棉根际卡那霉素抗性细菌群落多样性指数高于转基因棉,更均匀稳定。未发现转基因棉卡那霉素抗性标记基因nptII向根际细菌的基因漂移。

Dynamics in Kanamycin-Resistant Bacterial Population and Detection of npt Ⅱ Gene Flow in the Rhizosphere of Insect-Resistant Transgenic Cotton

【Objective】 The dynamics in kanamycin-resistant bacterial population were studied and the flow of npt II in the rhizosphere of insect-resistant transgenic cotton was monitored. 【Method】 Insect-resistant transgenic cotton GK1, GK19, 33B, and SGK3, and corresponding receptor cotton 33, Shiyuan 321 and Simian No.3 cotton were sampled at different growing stages. Dynamics and diversity among kanamycin-resistant bacterial populations in the rhizosphere were analyzed with the conventional culture methods. In addition, the flow of npt II was monitored by germ PCR, and the unique primer was designed from the sequence of npt II, the CK was pBI121 plasmids. 【Result】 The population of kanamycin-resistant bacteria in cotton rhizosphere decreased along with the cotton growth. Significant difference was found among the same cotton cultivar at different sample times, but little difference was found between insect-resistant transgenic cotton and corresponding receptor cotton at the same time. Simpson diversity index, Shannon-Wiener diversity index and Pielou’s evenness index were calculated, and the results showed that the diversity index of kanamycin-resistant bacterial population in rhizosphere of conventional cotton was larger than that of insect-resistant transgenic cotton. The monitoring result by PCR amplication showed that positive PCR products of 18 strains were found from 21 kanamycin-resistant strains, no sequences had 100% similarity with the known nptⅡ gene sequence. 【Conclusion】 It was suggested that little differences were found among the total rhizospheric population of kanamycin-resistant bacteria between insect-resistant transgenic cotton lines and their receptor cotton cultivars, the diversity index of rhizospheric kanamycin-resistant bacteria population of receptor cotton cultivar is larger than the corresponding insect-resistant transgenic cotton line, and is more even and stable. No gene flow of nptⅡ in insect-resistant transgenic cottons into the rhizosphere bacteria was detected.