马铃薯甲虫RNA干扰高效致死基因的筛选

Screening of Highly Effective Lethal Genes Interfered with RNA in Leptinotarsa decemlineata and Study on the Evaluation Indexes of Its Lethal Factors

【目的】基于备选基因RNA干扰后的致死效果评价,筛选出高效致死基因。【方法】通过在赤拟谷盗中已的报导和相似性搜索获得马铃薯甲虫致死基因Ldpp1alpha-96a、LdRpt3、Ldalpha snap、LdSrp54k、LdRpn6、LdRop、LdGawky、Ldshi、Ldcact、Ldinr-a,以及阳性对照基因LdATPaseA和LdATPaseE,构建dsRNA表达载体,经饲喂马铃薯甲虫二龄幼虫不同浓度的dsRNA,分析对幼虫的20%取食剂量AD 20、化蛹中量PD 50和幼虫7 d致死中量LD 50,经分析和评估,筛选出高效致死基因。【结果】12个基因的dsRNA对马铃薯甲虫幼虫的AD 20在1.18±0.10(μg/mL)~61.07±10.17(μg/mL);PD 50在0.06±0.01(μg/mL)~31.20±1.89(μg/mL);LD 50在1.39±0.13(μg/mL)~228.13±6.72(μg/mL)。其中AD 20最高与最低的基因分别为Ldinr-a、LdRpn6;PD 50最高与最低的基因分别为Ldinr-a、LdGawky;LD 0最高与最低的基因分别为Ldinr-a、Ldsrp54k。【结论】12个基因的dsRNA对马铃薯甲虫幼虫均有一定的抑制取食、抑制化蛹及致死活性。其致死活性随浓度增高而增大,表现出剂量依赖效应,与对照组存在显著差异。通过综合比较AD 20和LD 50,Ldalpha snap、LdRpn6、LdSrp54k、LdRpt3个基因的毒力较ATPaseE和ATPaseA更强,具有较高的生物活性。提出并推荐AD 20和LD 50是评价备选基因dsRNA对幼虫致死效果的关键指标。

【Objective】To screen out highly effective lethal genes based on evaluation of lethal effect of alternative gene RNA interference.【Method】Ten dsRNA expression vectors of Ldpp1alpha-96a,LdRpt3,Ldalpha snap,LdSrp54k,LdRpn6,LdRop,LdGawky,Ldshi,Ldcact,Ldinr-a and 2 positive control genes LdATPaseA and LdATPaseE were constructed,dsRNA were fed to the second instar larvae of Colorado Potato beetle with gradient concentrations,which was used to analyze AD 20,20%antifeedant dose,PD 50,medium pupation dose,and LD 50,medium lethal dose,to screen highly effective lethal genes and evaluate lethal efficiency.【Result】The results showed that:AD 20 of the above 12 genes for the larvae of potato beetle ranged from 1.18±0.10(μg/mL)-61.07±10.17(μg/mL);the PD 50 ranged from 0.06±0.01(μg/mL)-31.20±1.89(μg/mL);and the AD 20 ranged from 1.39±0.13(μg/mL)-228.13±6.72(μg/mL).The highest and lowest genes of AD 20 were Ldinr-a and LdRpn6,the highest and lowest genes of PD 50 were Ldinr-a and LdGawky,and the highest and lowest genes of LD 50 were Ldinr-a and Ldsrp54k,respectively.【Conclusion】All of the 12 alternative genes have an effect on inhibition of feeding,pupation and lethal activity.Their lethal activity increased with the increase of concentration,showing a dose-dependent effect,which was significantly different from the control group.By comparison of AD 20 and LD 50,5 genes of Ldcact,Ldalpha-snap,LdRpn6,LdSrp54k and LdRpt3 are the most effective.It is suggested and recommended that AD 20 and LD 50 are the key indexes to evaluate the lethal effect of alternative genes dsRNA on larvae.