中华蜜蜂气味受体基因AcerOr1 RNA最佳干扰片段的筛选

Construction of RNA interference plasmid targeting of AcerOr1 and optimal interference efficiency determination

为了更好地筛选出中华蜜蜂气味受体基因AcerOr1有效干扰序列,本研究以中华蜜蜂气味受体基因AcerOr1为靶标,分别选取3个片段AcerOr1-1(429 bp)、AcerOr1-2(218 bp)和AcerOr1-3(543 bp),并构建相应的dsRNA表达载体,获取dsRNA后通过单只饲喂进行体内RNAi实验,运用qPCR和Western blot从mRNA水平和蛋白水平检测干扰效率。实验结果显示:片段大小429 bp组ds AcerOr1-1和对照组mRNA表达量之间不具有显著的统计学差异(P>0.05),片段大小为218 bp的ds AcerOr1-2组和片段大小为543 bp的ds AcerOr1-3组对AcerOr1表达量都有显著的抑制(P<0.05),抑制率分别为88.85%±0.12%和69.16%±1.06%,以片段大小为218 bp的ds AcerOr1-2干扰效果最佳。Western blot结果显示:与mRNA表达水平检测结果一致,饲喂ds AcerOr1-1 AcerOr1蛋白的表达量与对照组差异不显著(P>0.05),而饲喂ds AcerOr1-2和ds AcerOr1-3后AcerOr1蛋白的表达量均受到显著抑制(P<0.05)。本试验成功构建AcerOr1基因干扰载体,最终确定218 bp的ds AcerOr1-2为最佳干扰片段,证实其能有效抑制目的基因mRNA和蛋白的表达,为进一步研究该基因的体内外功能奠定了基础。

In order to better screen out an effective interference sequence of AcerOr1,in this research,the olfactor receptors AcerOr1 of Apis cerana cerana was used as the target.Three fragments of AcerOr1-1(429 bp),AcerOr1-2(218 bp)and AcerOr1-3(543 bp)were selected,respectively,and the corresponding dsRNA expression vectors were constructed.After dsRNA was abtained,RNAi experiment was carried out in vivo through single feeding.qPCR and Western blot were used to detect interference efficiency from mRNA and protein levels.The results showed that there was no significant statistical difference in the mRNA expression of AcerOr1 between the ds AcerOr1-1 group with 429 bp fragment and the control group(P>0.05).The expression of AcerOr1 was significantly inhibited in ds AcerOr1-2 group with 218 bp fragment and ds AcerOr1-3 group with 543 bp fragment,and the inhibition rates were 88.85%±0.12%and 69.16%±1.06%,respectively(P<0.05).The ds AcerOr1-2 with the fragment of 218 bp had the best interference effect.The results of Western blot showed that the expression of AcerOr1 protein was not significantly difference of ds AcerOr1-1 group compared with the control group(P>0.05),while the expression of AcerOr1 protein was significantly inhibited after feeding ds AcerOr1-2 and ds AcerOr1-3(P<0.05).In this study,AcerOr1 gene interference vector was successfully constructed,and finally the ds AcerOr1-2 was selected as the best interference fragment,which effectively inhibit the expression of target gene mRNA and protein,laying a foundation for further study of the function of AcerOr1 in vivo and in vitro.