利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因

doi:10.3724/SP.J.1005.2014.0574

遗传 ›› 2014, Vol. 36 ›› Issue (6): 574-583.doi: 10.3724/SP.J.1005.2014.0574

利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因

曹振龙¹, 郝江叶¹, 周艳芬^{1, 2}, 张喆³, 倪志华¹, 胡远想³, 刘伟丽³, 李永超⁴, 马润林⁴, 王振山^{1, 2}

1. 河北大学生命科学学院, 保定 071002;
2. 河北省生物工程技术研究中心, 保定 071002;
3. 河北大学基础医学院, 保定 071002;
4. 中国科学院遗传与发育生物学研究所, 北京 100101; 5. Department of Pharmacology, University of Washington, Seattle, WA 98195

收稿日期:2013-11-24 修回日期:2014-01-16 出版日期:2014-06-20 发布日期:2014-05-28
通讯作者: 王振山,教授,博士生导师,研究方向：动物行为遗传分子机制。E-mail:zswang@hbu.edu.cn E-mail:dafeizizhu@163.com
作者简介:曹振龙,硕士研究生,专业方向：细胞分子生物学。E-mail:dafeizizhu@163.com
基金资助:
国家自然科学基金项目(编号：31171191), 河北省自然科学基金项目(编号：C2012201106), 教育部留学人员回国启动基金项目(教外司留<2013>693号)和河北省生物工程重点学科经费资助

Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach

Zhenlong Cao¹, Jiangye Hao¹, Yanfen Zhou^{1, 2}, Zhe Zhang³, Zhihua Ni¹, Yuanxiang Hu³, Weili Liu³, Yongchao Li⁴, Daniel R. Storm⁵, Runlin Z. Ma⁴, Zhenshan Wang^{1, 2}

1. College of Life Science, Hebei University, Baoding 071002, China;;
2. Research Center of Bioengineering of Hebei Province, Baoding 071002, China;;
3. College of Basic Medicine, Hebei University, Baoding 071002, China;;
4. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China;; 5. Department of Pharmacology, University of Washington, Seattle, WA 98195, USA

Received:2013-11-24 Revised:2014-01-16 Online:2014-06-20 Published:2014-05-28

摘要/Abstract

摘要：

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。

关键词: 腺苷酸环化酶3, 主要嗅觉表皮, 差异表达基因, 抑制性消减杂交

Abstract:

Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (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 hybridization (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 molecular 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 significantly, 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 speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.

Key words: adenylate cyclase 3 (), main olfactory epithelium (MOE), differentially expressed genes, suppression subtractive hybridization (SSH)

曹振龙, 郝江叶, 周艳芬, 张喆, 倪志华, 胡远想, 刘伟丽, 李永超, Daniel R. Storm, 马润林, 王振山. 利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因[J]. 遗传, 2014, 36(6): 574-583.

Zhenlong Cao, Jiangye Hao, Yanfen Zhou, Zhe Zhang, Zhihua Ni, Yuanxiang Hu, Weili Liu, Yongchao Li, Daniel R. Storm, Runlin Z. Ma, Zhenshan Wang. Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach[J]. HEREDITAS(Beijing), 2014, 36(6): 574-583.

参考文献

[1]Restrepo D, Arellano J, Oliva AM, Schaefer ML, Lin W. Emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice. Horm Behav, 2004, 46(3): 247-256.
[2]Wang ZS, Nudelman A, Storm DR. Are pheromones detected through the main olfactory epithelium? Mol Neurobiol, 2007, 35(3): 317-323.
[3]Belluscio L, Gold GH, Nemes A, Axel R. Mice deficient in G(olf) are anosmic. Neuron, 1998, 20(1): 69-81.
[4]Wong ST, Trinh K, Hacker B, Chan GC, Lowe G, Gaggar A, Xia Z, Gold GH, Storm DR. Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice. Neuron, 2000, 27(3): 487- 497.
[5]Brunet LJ, Gold GH, Ngai J. General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide- gated cation channel. Neuron, 1996, 17(4): 681-693.
[6]Chen XM, Xia ZG, Storm DR. Stimulation of electro- olfactogram responses in the main olfactory epithelia by airflow depends on the type 3 adenylyl cyclase. J Neurosci, 2012, 32(45): 15769-15778.
[7]Wang ZS, Li V, Chan GCK, Phan T, Nudelman AS, Xia ZQ, Storm DR. Adult type 3 adenylyl cyclase-deficient mice are obese. PLoS One, 2009, 4(9): e6979.
[8]Wang ZS, Storm DR. Maternal behavior is impaired in female mice lacking type 3 adenylyl cyclase. Neuropsy-chopharmacology, 2010, 36(4): 772-781.
[9]Wang ZS, Phan T, Storm DR. The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory: role of cAMP signaling in primary cilia. J Neurosci, 2011, 31(15): 5557-5561.
[10]Zhang XM, Firestein S. The olfactory receptor gene superfamily of the mouse. Nat Neurosci, 2002, 5(2): 124- 133.
[11]Diatchenko L, Lukyanov S, Lau YFC, Siebert PD. Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol, 1999, 303: 349-380.
[12]Wang ZS, Storm DR. Extraction of DNA from mouse tails. Biotechniques, 2006, 41(4): 410, 412.
[13]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25(4): 402-408.
[14]Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93(12): 6025-6030.
[15]Wang ZS, Farmer K, Hill GE, Edwards SV. A cDNA macroarray approach to parasite-induced gene expression changes in a songbird host: genetic response of house finches to experimental infection by Mycoplasma galli-sep-ticum. Mol Ecol, 2006, 15(5): 1263-1273.
[16]Saban MR, Hellmich HL, Simpson C, Davis CA, Lang ML, Ihnat MA, O'Donnell MA, Wu XR, Saban R. Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single- spanning uroplakins. BMC Cancer, 2007, 7: 204.
[17]Sahebi M, Hanafi MM, Abdullah SNA, Rafii MY, Azizi P, Nejat N, Idris AS. Isolation and expression analysis of novel silicon absorption gene from roots of mangrove (Rhizophora apiculata) via suppression subtractive hybridiza-tion. Biomed Res Int, 2014, 2014: 971-985.
[18]Ni YH, Ji CB, Wang B, Qiu J, Wang JW, Guo XR. A Novel pro-adipogenesis factor abundant in adipose tissues and over-expressed in obesity acts upstream of PPARgamma and C/EBPalpha. J Bioenerg Biomembr, 2013, 45(3): 219- 228.
[19]Mottaghi-Dastjerdi N, Soltany-Rezaee-Rad M, Sepehrizadeh Z, Roshandel G, Ebr

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因

Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 9

编辑推荐

Metrics

[1]	郭彦, 杨乐乐, 戚华宇. 小鼠雄性生殖干细胞转录组分析揭示成熟精原干细胞特征[J]. 遗传, 2022, 44(7): 591-608.
[2]	陈家辉, 任学义, 李丽敏, 卢诗意, 程湉, 谭量天, 梁少东, 何丹林, 罗庆斌, 聂庆华, 张细权, 罗文. 转录组测序揭示细胞周期通路参与鸡腹脂沉积[J]. 遗传, 2019, 41(10): 962-973.
[3]	陈昊蒋桂雄龙洪旭谭晓风. 基于油桐种子3个不同发育时期转录组的油脂合成代谢途径分析[J]. 遗传, 2013, 35(12): 1403-1414.
[4]	杨立伟，施季森. 不同浓度外源IAA处理对杉木茎部基因表达的影响[J]. 遗传, 2012, 34(4): 472-484.
[5]	田万年，张守发，李香子，高青山，金鑫，严昌国. 延边黄牛背最长肌差异表达基因的筛选、克隆及序列分析[J]. 遗传, 2011, 33(11): 1219-1224.
[6]	张蕾，齐力旺，韩素英 . 落叶松体细胞胚成熟阶段差异表达的基因及部分基因的表达谱分析[J]. 遗传, 2009, 31(5): 540-545.
[7]	刘关君，刘明坤，许志茹，阎秀峰，魏志刚，杨传平. 盐胁迫下西伯利亚蓼茎叶正反消减文库的构建及初步分析[J]. 遗传, 2009, 31(4): 426-433.
[8]	袁晓萌，周云涛，张红岩，薛华，周琳，赵云. 甘蓝型油菜小核糖核蛋白BnSmD1编码区全长cDNA 的克隆与表达分析[J]. 遗传, 2007, 29(12): 1525-1528.
[9]	李玉昌，徐存拴，张云汉. 应用抑制性消减杂交技术克隆大鼠肝再生过程中特异表达基因[J]. 遗传, 2002, 24(2): 152-154.