基于转录组测序筛选不同性别C57BL/6J小鼠差异基因及通路

Screening of differential genes and pathways in C57BL/6J mice of different genders based on transcriptome sequencing

目的通过转录组测序筛选雌性与雄性C57BL/6J小鼠差异基因及通路,为进一步探讨小鼠雌雄间行为学差异的分子机制奠定基础。方法C57BL/6J品系的8周龄雌性与雄性小鼠,完整分离小鼠海马体组织,提取总RNA后逆转录构建cDNA文库,并在华大基因平台上生成单端为50个碱基的读数进行转录组测序。测序结束后基于GO和KEGG数据库,结合R语言中的phyper函数对数据进行筛选、校正和富集分析,计算P值;然后对P值进行矫正得到Q值并基于超几何测试方法对带注释的不同基因表达情况进行了分析,筛选出不同性别小鼠海马体中的差异基因及通路。测序结束后,使用实时荧光定量PCR(RT-qPCR)验证在雌雄小鼠海马中筛选出的差异基因并比较差异基因在不同信号通路上的表达情况。结果通过雌雄差异比较,筛选出325个差异基因,其中上调基因233个,下调基因92个,这些差异基因功能主要富集于长时程增强(LTP)、钙离子信号通路、尼古丁成瘾等过程;雌雄差异基因相互作用网络共有362个连接点和1703个相互作用边,筛选出的10个核心基因分别为:赖氨酸脱甲基酶5D(Kdm5d)、细胞周期蛋白依赖性激酶5(Cdkl5)、细胞输出介质(Cle1)、多巴胺受体6a3(Slc6a3)、盒式转录因子(Fox)、前体mRNA加工因子4B(Prpf4b)、甘氨酸受体4(Glur4)、钙离子受体2(Camk2)、DNA和RNA结合蛋白家族(Son)、5-羟色胺受体5b(Htr5b)。RT-qPCR验证结果与测序结果一致。结论从雌雄小鼠海马体中筛选出差异基因为研究不同性别小鼠行为学差异提供实验思路并为后续小鼠行为学研究做出针对性实验设计。

Objective To screen the differential genes and pathways of female and male C57BL/6J mice by transcriptome sequencing,and to lay a foundation for further exploring the molecular mechanism of behavioral differences between male and female mice.Methods 8-week-old female and male mice of the C57BL/6J strain were completely isolated from the mouse hippocampus,and total RNA was extracted and reverse transcribed to construct a cDNA library,and 50 single-ended mice were generated on the BGIseq500 platform(BGl-Shenzhen,China)The base reads were transcriptome sequenced.After sequencing,based on GO and KEGG databases,combined with the phyper function in R language to screen,correct and enrich the data,calculate P value,and then perform FDR correction on P value to obtain Q value and analyze different annotated genes based on the hypergeometric test method.The expression status was analyzed,and the differential genes and pathways in the hippocampus of mice of different genders were screened out.Results Through the comparison of male and female differences,325 differential genes were screened,including 233 up-regulated genes and 92 down-regulated genes.The functions of these differential genes were mainly enriched in long-term potentiation(LTP),calcium signal pathway,nicotine addiction and other processes.There were 362 junctions and 1703 interaction edges in the female-male differential gene interaction network,and 10 core genes selected:lysine demethylase 5D(Kdm5d),cyclin-dependent kinase 5(Cdkl5),Cell output mediator(Cle1),dopamine receptor 6a3(Slc6a3),cassette transcription factor(Fox),precursor mRNA processing factor 4B(Prpf4b),glycine receptor 4(Glur4),calcium ion receptor 2(Camk2),DNA and RNA binding protein family(Son),serotonin receptor 5b(Htr5b).Conclusion The selected differential genes and signal pathways may lay the foundation for explaining the molecular mechanism of the differences in behavior between male and female mice.