CAJ | 학술논문

腺苷酸环化酶3(Adenylate cyclase 3, AC3)基因在小鼠主要嗅觉表皮(Main olfactory epithelium, MOE)内的嗅觉信号传导中起着重要作用, AC3缺失是否会导致MOE内与之相关的基因发生差异表达,尚待确定。文章利用抑制性消减杂交(Suppression subtractive hybridization, SSH)方法,以AC3敲除(AC3-/-)及其同窝出生的野生型(AC3+/+)小鼠 MOE为材料,构建了正向和反向两个消减文库,采用斑点杂交对消减文库进行初步筛选,对筛选出的差异表达基因进行序列测定及生物信息学分析,并利用荧光定量PCR(qRT-PCR)方法对其进行验证。斑点杂交筛选获得了386个差异表达克隆,随机选取其中的80个进行DNA序列测定,经序列比对后发现有62个在GenBank上获得了与之相匹配的基因信息,其中24个上调差异表达克隆对应于kcnk3、mapk7、megf11等基因,38个下调差异表达克隆对应于tmem88b、c-mip、skp1a、mlycd等基因。利用Gene Ontology(GO)方法对这些差异表达基因进行蛋白功能注释,发现它们主要集中在分子结合、细胞周期、生物和细胞过程等功能方面。选取其中上调基因 kcnk3和下调基因 c-mip、mlycd、tmem88b 及 trappc5进行 qRT-PCR 验证。结果表明,在 AC3-/-小鼠MOE内kcnk3的表达量显著上调,是对照组小鼠的1.27倍,而c-mip、mlycd、tmem88b和trappc5的表达量显著下调,为对照组小鼠的20%、7%、32%和29%。这些基因的功能与 K+通道、细胞发育与分化、脂肪代谢和膜蛋白转运等密切相关。推测它们可能与AC3基因共同作用,调节小鼠MOE内的嗅觉信号传导信息。
선감산배화매3(Adenylate cyclase 3, AC3)기인재소서주요후각표피(Main olfactory epithelium, MOE)내적후각신호전도중기착중요작용, AC3결실시부회도치MOE내여지상관적기인발생차이표체,상대학정。문장이용억제성소감잡교(Suppression subtractive hybridization, SSH)방법,이AC3고제(AC3-/-)급기동와출생적야생형(AC3+/+)소서 MOE위재료,구건료정향화반향량개소감문고,채용반점잡교대소감문고진행초보사선,대사선출적차이표체기인진행서렬측정급생물신식학분석,병이용형광정량PCR(qRT-PCR)방법대기진행험증。반점잡교사선획득료386개차이표체극륭,수궤선취기중적80개진행DNA서렬측정,경서렬비대후발현유62개재GenBank상획득료여지상필배적기인신식,기중24개상조차이표체극륭대응우kcnk3、mapk7、megf11등기인,38개하조차이표체극륭대응우tmem88b、c-mip、skp1a、mlycd등기인。이용Gene Ontology(GO)방법대저사차이표체기인진행단백공능주석,발현타문주요집중재분자결합、세포주기、생물화세포과정등공능방면。선취기중상조기인 kcnk3화하조기인 c-mip、mlycd、tmem88b 급 trappc5진행 qRT-PCR 험증。결과표명,재 AC3-/-소서MOE내kcnk3적표체량현저상조,시대조조소서적1.27배,이c-mip、mlycd、tmem88b화trappc5적표체량현저하조,위대조조소서적20%、7%、32%화29%。저사기인적공능여 K+통도、세포발육여분화、지방대사화막단백전운등밀절상관。추측타문가능여AC3기인공동작용,조절소서MOE내적후각신호전도신식。
Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epi-thelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybri-dization (SSH) approach, with MOEs from AC3-/-and AC3+/+mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on mo-lecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3+/+and AC3-/-mice. The data indicate that kcnk3 gene is up-regulated signifi-cantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3+/+mice. The functions of these genes are closely related with K+channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to spe-culate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.