中华蜜蜂气味受体AcerOr2基因最佳干扰片段的筛选

Screening of Best Interference Fragment of Apis cerana cerana Olfactor Receptors AcerOr2 Gene

为了筛选出中华蜜蜂气味受体基因AcerOr2最佳干扰效率的序列,以中华蜜蜂气味受体AcerOr2基因为靶标,分别选取3个片段AcerOr2-1(411 bp)、AcerOr2-2(205 bp)和AcerOr2-3(471 bp),并构建相应的dsRNA表达载体,获取dsRNA后进行RNAi试验,通过qPCR和Western从mRNA水平和蛋白水平检测干扰效率。mRNA结果显示,饲喂3个不同的片段AcerOr2-1(411 bp)、AcerOr2-2(205 bp)和AcerOr2-3(471 bp)对AcerOr2表达量均会产生抑制作用,抑制率分别为69.72%±0.49%、47.26%±0.25%、73.17%±0.97%,抑制率最佳的是471 bp的dsRNA AcerOr2-3。Western Blot结果显示,饲喂3个大小不同的干扰片段后AcerOr2蛋白的表达量均受到显著抑制。试验成功构建AcerOr2基因干扰载体,最终确定471 bp的dsRNA AcerOr2-3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。研究结果可为进一步应用RNAi技术研究该基因的体内外功能奠定基础。

To screen out the sequence of the best interference efficiency of AcerOr2 of Apis cerana cerana,in this research,the olfactor receptors AcerOr2 of Apis cerana cerana was used as the target,three fragments of AcerOr2-1(411 bp),AcerOr2-2(205 bp)and AcerOr2-3(471 bp)were selected,respectively,and the corresponding dsRNA expression vectors were constructed based on the target of olfactor receptors AcerOr2 of Apis cerana cerana.After obtaining dsRNA,RNAi experiment was carried out and the interference efficiency was detected from mRNA level and protein level by qPCR and Western.The results of mRNA showed that three different fragments of AcerOr2-1(411 bp),AcerOr2-2(205 bp)and AcerOr2-3(471 bp)could inhibit the expression of AcerOr2,and the inhibition rates were 69.72%±0.49%,47.26%±0.25%and 73.17%±0.97%,respectively.The dsRNA AcerOr2-3 interference effect with fragment size of 471 bp was the best.The results of Western Blot showed that the expression of AcerOr2 protein was significantly inhibited after feeding three interfering fragments of different sizes.In this experiment,AcerOr2 gene interference vector was successfully constructed,and finally the dsRNA AcerOr2-3 of 471 bp was determined as the best interference fragment,which confirmed that it could effectively inhibit the expression of target gene mRNA and protein.The research results will lay a foundation for further study of function of the gene in vivo and in vitro by RNAi.