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, Ebrahimifard F, Setayesh N. Genome expression analysis by suppression subtractive hybridiza-tion identified overexpression of Humanin, a target gene in gastric cancer chemoresistance. Daru, 2014, 22(1): 14.
[20]Xu W, Li H, Wang R, Lei Z, Mao YQ, Wang X, Zhang YZ, Guo TT, Song RJ, Zhang XJ, Jin L, Li ZX, Irwin DM, Niu G, Tan HR. Differential expression of genes associated with the progression of renal disease in the kidneys of liver-specific glucokinase gene knockout mice. Int J Mol Sci, 2013, 14(3): 6467-6486.
[21]Wang R, Gao H, Xu W, Li H, Mao YQ, Wang Y, Guo TT, Wang X, Song RJ, Li ZX, Irwin DM, Niu G, Tan HR. Differential expression of genes and changes in glucose metabolism in the liver of liver-specific glucokinase gene knockout mice. Gene, 2013, 516(2): 248-254.
[22]Ren HJ, Cui J, Yang W, Liu RD, Wang ZQ. Identification of differentially expressed genes of Trichinella spiralis larvae after exposure to host intestine milieu. PLoS ONE, 2013, 8(6): e67570.
[23]Goldstein SA, Bockenhauer D, O'Kelly I, Zilberberg N. Potassium leak channels and the KCNK family of two-P- domain subunits. Nat Rev Neurosci, 2001, 2(3): 175-184.
[24]Zhou G, Bao ZQ, Dixon JE. Components of a new human protein kinase signal transduction pathway. J Biol Chem, 1995, 270(21): 12665-12669.
[25]Fishman-Jacob T, Reznichenko L, Youdim MB, Mandel SA. A sporadic Parkinson disease model via silencing of the ubiquitin-proteasome/E3 ligase component SKP1A. J Biol Chem, 2009, 284(47): 32835-32845.
[26]Gabriel L, Lvov A, Orthodoxou D, Rittenhouse AR, Kobertz WR, Melikian HE. The acid-sensitive, anesthetic- activated potassium leak channel, KCNK3, is regulated by 14-3-3beta-dependent, protein kinase C (PKC)-mediated endocytic trafficking. J Biol Chem, 2012, 287(39): 32354- 32366.
[27]Scrivens PJ, Noueihed B, Shahrzad N, Hul S, Brunet S, Sacher M. C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking. Mol Biol Cell, 2011, 22(12): 2083-2093.
[28]Talley EM, Solorzano G, Lei Q, Kim D, Bayliss DA. Cns distribution of members of the two-pore-domain (KCNK) potassium channel family. J Neurosci, 2001, 21(19): 7491- 7505.
[29]Zuzarte M, Heusser K, Renigunta V, Schlichthörl G, Rinne S, Wischmeyer E, Daut J, Schwappach B, Preisig-Müller R. Intracellular traffic of the K+ channels TASK-1 and TASK-3: role of N- and C-terminal sorting signals and interaction with 14-3-3 proteins. J Physiol, 2009, 587(5): 929-952.
[30]Meyer DK, Fischer C, Becker U, Gottsching I, Boutillier S, Baermann C, Schmidt G, Klugbauer N, Leemhuis J. Pituitary adenylyl cyclase-activating polypeptide 38 reduces astroglial proliferation by inhibiting the GTPase RhoA. J Biol Chem, 2005, 280(26): 25258-25266.
[31]Grimbert P, Valanciute A, Audard V, Pawlak A, Le gouvelo S, Lang P, Niaudet P, Bensman A, Guellaën G, Sahali D. Truncation of C-mip (Tc-mip), a new proximal signaling protein, induces c-maf Th2 transcription factor and cytoskeleton reorganization. J Exp Med, 2003, 198(5): 797-807.
[32]Newbury DF, Winchester L, Addis L, Paracchini S, Buckingham LL, Clark A, Cohen W, Cowie H, Dworzynski K, Everitt A, Goodyer IM, Hennessy E, Kindley AD, Miller LL, Nasir J, O'Hare A, Shaw D, Simkin Z, Simonoff E, Slonims V, Watson J, Ragoussis J, Fisher SE, Seckl JR, Helms PJ, Bolton PF, Pickles A, Conti-Ramsden G, Baird G, Bishop DVM, Monaco AP. CMIP and ATP2C2 modulate phonological short-term memory in language impairment. Am J Hum Genet, 2009, 85(2): 264-272.
[33]Sendeyo K, Audard V, Zhang SY, Fan QF, Bouachi K, Ollero M, Rucker-Martin C, Gouadon E, Desvaux D, Bridoux F, Guellaën G, Ronco P, Lang P, Pawlak A, Sahali D. Upregulation of c-mip is closely related to podocyte dysfunction in membranous nephropathy. Kidney Int, 2013, 83(3): 414-425.
[34]Ory V, Fan QF, Hamdaoui N, Zhang SY, Desvaux D, Audard V, Candelier M, Noel LH, Lang P, Guellaen G, Pawlak A, Sahali D. C-mip down-regulates NF-kB activity and promotes apoptosis in podocytes. Am J Pathol, 2012, 180(6): 2284-2292.
[35]Kamal M, Pawlak A, BenMohamed F, Valanciute A, Dahan K, Candelier M, Lang P, Guellaën G, Sahali D. C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase. FEBS Lett, 2010, 584(3): 500- 506.
[36]Dyck JR, Barr AJ, Barr RL, Kolattukudy PE, Lopaschuk GD. Characterization of cardiac malonyl-CoA decarboxylase and its putative role in regulating fatty acid oxidation. Am J Physiol, 1998, 275(6 Pt 2): H2122-H2129.
[37]Kudo N, Barr AJ, Barr RL, Desai S, Lopaschuk GD. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5′-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem, 1995, 270(29): 17513-17520.
[38]Park H, Kaushik VK, Constant S, Prentki M, Przyby-tkowski E, Ruderman NB, Saha AK. Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-CoA carboxylase by AMP- activated protein kinase in rat tissues in response to exercise. J Biol Chem, 2002, 277(36): 32571-32577.
[39]Bennett MJ, Harthcock PA, Boriack RL, Cohen JC. Impaired mitochondrial fatty acid oxidative flux in fibro-blasts from a patient with malonyl-CoA decarboxylase deficiency. Mol Genet Metab, 2001, 73(3): 276-279.
[40]Rasche S, Toetter B, Adler J, Tschapek A, Doerner JF, Kurtenbach S, Hatt H, Meyer H, Warscheid B, Neuhaus EM. Tmem16b is specifically expressed in the cilia of olfactory sensory neurons. Chemical Senses, 2010, 35(3): 239-245.
[41]Lee HJ, Finkelstein D, Li X, Wu D, Shi DL, Zheng JJ. Identification of transmembrane protein 88 (TMEM88) as a dishevelled-binding protein. J Biol Chem, 2010, 285(53): 41549-41556.
[42]Stewart DJ. Wnt signaling pathway in non-small cell lung cancer. J Natl Cancer Inst, 2014, 106(1): djt356.
[43]Kummel D, Müller JJ, Roske Y, Misselwitz R, Büssow K, Heinemann U. The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex. EMBO Rep, 2005, 6(8): 787-793.
[44]Juilfs DM, Fülle HJ, Zhao AZ, Houslay MD, Garbers DL, Beavo JA. A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduc-tion pathway. Proc Natl Acad Sci USA, 1997, 94(7): 3388-3395.
[45]Chen S, Lane AP, Bock R, Leinders-Zufall T, Zufall F. Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors". J Neurophysiol, 2000, 84(1): 575-580

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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

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