转基因柔嫩艾美耳球虫对生态环境的影响

摘要

[目的]从表达M2e EYFP融合蛋白转基因柔嫩艾美耳球虫的稳定性,及其在环境中的传播能力、竞争能力和存活能力方面,分析转基因球虫对生态环境的潜在风险.[方法]稳定性试验包括:转基因球虫感染非靶动物,观察临床症状和卵囊产出情况;转基因球虫继代繁殖,检测荧光率;转基因球虫与毒害艾美耳球虫混合感染,PCR检测卵囊中的外源基因.传播能力试验主要是分析感染鸡和不感染同居鸡的卵囊产量和荧光率.竞争性试验则是用不同混合比例的2种球虫感染鸡,检测卵囊产量和荧光率;存活能力试验是用不同处理方式的卵囊感染鸡,检测卵囊是否产出以分析卵囊存活能力.[结果]转基因球虫不感染小鼠和家鸽,与野生型柔嫩艾美耳球虫宿主特异性相同;连续繁殖传17代后外源基因稳定表达.与毒害艾美耳球虫混合寄生时在异种球虫体内未检测出外源基因.传播能力试验中,46 d攻毒时转基因球虫感染与不感染同居组的卵囊产量差异不显著,但与不感染对照组相比均显著减少,且1∶1感染和不感染同居组卵囊荧光率检测结果维持在51.6％～67.9％.竞争性试验中,攻毒时各比例感染组卵囊产量均极显著少于不感染攻毒对照组,荧光率检测结果各比例组变化幅动不大,且1∶1组哨兵鸡卵囊荧光率检测结果为41.2％～61％.存活能力试验,转基因球虫与野生型球虫在夏季室温条件下均不能存活90 d,且与其他不同方式处理的卵囊外界存活时间接近.[结论]说明转基因球虫具有良好的稳定性,在环境中的传播能力、竞争能力和存活能力未呈现显著优于野生球虫的趋势.%[Objective]To preliminarily analyze the impact of transgenic E.tenella line expressing M2e-EYFP fusion protein on the ecological environments.the genetic stability,transmission ability,competitive and survival ability were determined.[Methods]The stability were evaluated by observing clinical symptoms and oocyst output in non-target animals infected transgenic coccidian oocysts;the fluorescence rate of coccidian oocysts in their subculture breeding and the exogenous gene in oocysts of chickens inoculated with the E.necatrix and the transgenic E.tenella were detected by PCR.Propagation ability test was to detect oocysts production and oocysts fluorescence rate in cohabiting chickens infected and non-infected coccidian oocysts.While,there were different treatments,non infected chickens were cohabited with infected chickens in transmission test,and chickens were infected with different mixed proportions of two species of coccidia in competitive test and the outputs and fluorescence rate of oocysts from their chickens.The detection of oocyst outputs from infected chickens with different treatments of oocysts was to evaluate the survival of oocysts.[Results]The transgenic coccidian had the same host specificity as wild-type E.tenella and didn't cause pathogenic changes to the mice and pigeon.The exogenous gene expression remained stable after continuously breeding 17 generations,and the exogenous gene was not detected in the heterologous coccidia when mixed with transgenic E.tenella.In propagative test,there was no significant difference in the oocyst output from inoculated with transgenic E.tenella chickens group and cohabitation group after challenged (46d),while it decreased significantly compared with non-inoculated and challenge groups.And the fluorescent ratio of progeny oocysts of chickens inoculated with mixed oocysts (1∶1) group and cohabitation group maintained 51.6 ％ ～ 67.9 ％.In competitive test,the oocyst output of each inoculated group was decreased significantly after challenge compared with non-inoculated and challenge groups and the fluorescent ratio of progeny oocysts of inoculated with mixed oocysts groups varied a little.Moreover,sentinel chickens were inoculated with transgenic E.tenella mixed with wild-type(1∶1),their oocyst fluorescence rate results showed in 41.2 ％-61 ％.[Conclusion]The results showed that transgenic E.tenella have no better propagative,competitive and survival ability than that of the wild type strain,which suggest that the impact of the transgenic E.tenella on the ecological environment is similar to wild-type.