Objective To conduct bioinformatics analysis of MyD88 gene of Anopheles stephensi and study its role in the anti-infection of Plasmodium yoelii.
Methods Firstly, MyD88 gene sequence was obtained from the VectorBase database, and bioinformatic analysis of MyD88 gene sequence was conducted using softwares such as VectorNTI and DNAMAN. Then, the primers for Real- time PCR were designed according to cDNA sequence of MyD88. Total RNA of Anopheles stephensi was extracted and cDNA was synthesized through reverse transcribe method. Finally, Real-time PCR was carried out to detect the transcriptional level of MyD88 gene in various life stages of mosquitoes and the changes after infection with Plasmodium yoelii.
Results MyD88 gene located on the site of 155 163-158 066 bp of antisense strand of chromosome KB664619 of Anopheles stephensi. It contained two exons with the length of cDNA of 1 383 bp and encodes 460 amino acids. Phylogenetic tree results showed that the phylogenetic distance of Anopheles stephensi was close to that of Anopheles dirus and Anopheles gambiae with a sequence homology of 79%. The result of transcriptional level study showed that the transcription of MyD88 gene of Anopheles stephensi was up-regulated by both normal and infected blood feeding. On the 3rd and 5th day post blood feeding, the expression of MyD88 gene was significantly up-regulated comparing with that in the normal blood feeding group and the sugar feeding group.
Conclusion This study confirmed that MyD88 gene played an important role in the innate immunity of mosquitoes against plasmodium infection in the Toll signaling pathway, and the effect was obvious 3 days after infection.
摘要： 目的 对斯氏按蚊 MyD88 基因进行生物信息学分析, 并研究其在抗约氏疟原虫感染中的作用。 方法 首 先, 通过VectorBase数据库获取斯氏按蚊 MyD88 基因cDNA序列, 并利用VectorNTI、DNAMAN等软件对 MyD88 基因cD- NA序列进行生物信息学分析。然后, 根据 MyD88 的cDNA序列设计Real-time PCR引物, 提取斯氏按蚊总RNA, 通过反 转录合成cDNA, 利用Real-time PCR方法检测 MyD88 基因在蚊各个生活史阶段转录水平情况及其在约氏疟原虫感染 后的转录水平变化。 结果 斯氏按蚊 MyD88 基因位于斯氏按蚊KB664619号染色体155 163~158 066 bp的反义链上, 包含两个外显子, 其cDNA全长1 383 bp, 编码460个氨基酸。系统发育树结果显示, 斯氏按蚊与大劣按蚊和冈比亚按 蚊系统发育的距离近, 其氨基酸序列同源性达79%。对斯氏按蚊各时期 MyD88 基因转录水平的研究结果显示, 吸食正 常血和感染血均能够上调 MyD88 的转录, 吸血后3 d和5 d吸感染血组 MyD88 基因转录水平上调明显, 显著高于正常血 组和未吸血组。 结论 本研究证实了Toll信号通路中的 MyD88 基因在蚊抗疟原虫感染的天然免疫中发挥了重要作用, 在感染后3 d时作用明显。