曹振龍, 郝江葉, 周艷芬,2, 張喆, 倪志華, 胡遠(yuǎn)想, 劉偉麗, 李永超, Daniel R. Storm, 馬潤林, 王振山,2

1. 河北大學(xué)生命科學(xué)學(xué)院, 保定 071002;

2. 河北省生物工程技術(shù)研究中心, 保定 071002;

3. 河北大學(xué)基礎(chǔ)醫(yī)學(xué)院, 保定 071002;

4. 中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所, 北京 100101;

5. Department of Pharmacology, University of Washington, Seattle, WA 98195

利用SSH方法篩選與鑒定AC3基因缺失小鼠主要嗅覺表皮內(nèi)的差異表達(dá)基因

曹振龍1, 郝江葉1, 周艷芬1,2, 張喆3, 倪志華1, 胡遠(yuǎn)想3, 劉偉麗3, 李永超4, Daniel R. Storm5, 馬潤林4, 王振山1,2

1. 河北大學(xué)生命科學(xué)學(xué)院, 保定 071002;

2. 河北省生物工程技術(shù)研究中心, 保定 071002;

3. 河北大學(xué)基礎(chǔ)醫(yī)學(xué)院, 保定 071002;

4. 中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所, 北京 100101;

5. Department of Pharmacology, University of Washington, Seattle, WA 98195

腺苷酸環(huán)化酶3(Adenylate cyclase 3, AC3)基因在小鼠主要嗅覺表皮(Main olfactory epithelium, MOE)內(nèi)的嗅覺信號傳導(dǎo)中起著重要作用, AC3缺失是否會導(dǎo)致MOE內(nèi)與之相關(guān)的基因發(fā)生差異表達(dá), 尚待確定。文章利用抑制性消減雜交(Suppression subtractive hybridization, SSH)方法, 以AC3敲除(AC3-/-)及其同窩出生的野生型(AC3+/+)小鼠MOE為材料, 構(gòu)建了正向和反向兩個消減文庫, 采用斑點雜交對消減文庫進(jìn)行初步篩選, 對篩選出的差異表達(dá)基因進(jìn)行序列測定及生物信息學(xué)分析, 并利用熒光定量PCR(qRT-PCR)方法對其進(jìn)行驗證。斑點雜交篩選獲得了386個差異表達(dá)克隆, 隨機選取其中的80個進(jìn)行DNA序列測定, 經(jīng)序列比對后發(fā)現(xiàn)有62個在GenBank上獲得了與之相匹配的基因信息, 其中24個上調(diào)差異表達(dá)克隆對應(yīng)于kcnk3、mapk7、megf11等基因, 38個下調(diào)差異表達(dá)克隆對應(yīng)于tmem88b、c-mip、skp1a、mlycd等基因。利用Gene Ontology(GO)方法對這些差異表達(dá)基因進(jìn)行蛋白功能注釋, 發(fā)現(xiàn)它們主要集中在分子結(jié)合、細(xì)胞周期、生物和細(xì)胞過程等功能方面。選取其中上調(diào)基因kcnk3和下調(diào)基因c-mip、mlycd、tmem88b及trappc5進(jìn)行qRT-PCR驗證。結(jié)果表明, 在AC3-/-小鼠MOE內(nèi)kcnk3的表達(dá)量顯著上調(diào), 是對照組小鼠的1.27倍, 而c-mip、mlycd、tmem88b和trappc5的表達(dá)量顯著下調(diào), 為對照組小鼠的20%、7%、32%和29%。這些基因的功能與K+通道、細(xì)胞發(fā)育與分化、脂肪代謝和膜蛋白轉(zhuǎn)運等密切相關(guān)。推測它們可能與AC3基因共同作用, 調(diào)節(jié)小鼠MOE內(nèi)的嗅覺信號傳導(dǎo)信息。

腺苷酸環(huán)化酶3; 主要嗅覺表皮; 差異表達(dá)基因; 抑制性消減雜交

嗅覺在動物乃至人類的生命活動中具有重要作用。哺乳動物的嗅覺感知是由兩個解剖和功能上相互獨立的器官介導(dǎo)的, 即主要嗅覺表皮(Main olfactory epithelium, MOE)和犁鼻器 (Vomeronasal organ, VNO)。MOE的主要功能是感受揮發(fā)性化學(xué)信號, 識別種內(nèi)不同個體的社會地位; VNO的主要功能是感受信息素, 控制生殖及其相關(guān)行為[1,2]。單磷酸環(huán)化腺苷酸(cAMP)通路是MOE組織內(nèi)主要的嗅覺信號傳導(dǎo)通路, 由 Golf蛋白[3]、腺苷酸環(huán)化酶 3(AC3)[4]和環(huán)核苷酸門控離子通道(CNG channel)[5]等組成。研究發(fā)現(xiàn), 敲除AC3基因的小鼠(AC3-/-)嗅覺功能喪失[4], 并且在雄性攻擊、母性關(guān)愛、體重、學(xué)習(xí)與記憶和生殖能力等諸多方面均受到影響[6～9]。

小鼠基因組內(nèi)有 1000個以上的嗅覺受體基因(Olfactory receptor, OR)[10]表達(dá)于MOE組織內(nèi)的嗅覺神經(jīng)細(xì)胞, 絕大多數(shù)嗅覺神經(jīng)細(xì)胞使用單一cAMP信號傳導(dǎo)通路來感知氣味分子。生物體內(nèi)每一個基因的表達(dá)都是網(wǎng)絡(luò)調(diào)控的結(jié)果。在MOE組織內(nèi), 敲除AC3基因后是否會導(dǎo)致相關(guān)基因表達(dá)發(fā)生改變, 尚未見報道。抑制性消減雜交技術(shù)(Suppression subtractive hybridization, SSH)是篩選差異表達(dá)基因的快捷有效方法[11], 被廣泛應(yīng)用于多個領(lǐng)域的研究。

本研究利用 SSH方法, 以來源于 AC3-/-和其同窩出生的野生型小鼠MOE組織為材料, 構(gòu)建了正向和反向消減雜交文庫, 運用斑點雜交、DNA測序、熒光定量 PCR(qRT-PCR)等技術(shù), 對文庫進(jìn)行初步篩選與鑒定, 篩選出多個在MOE組織內(nèi)受AC3調(diào)控的差異表達(dá)基因, 為進(jìn)一步深入了解嗅覺信號傳導(dǎo)機制奠定了初步基礎(chǔ)。

1 材料和方法

1.1 小鼠基因型鑒定、總RNA提取及cDNA合成

本研究所使用的 AC3+/-(C57BL/6J)小鼠從美國華盛頓大學(xué)(西雅圖分校)Daniel.R. Storm 實驗室引進(jìn), 并飼養(yǎng)于河北大學(xué)標(biāo)準(zhǔn)動物房。后代小鼠基因型按照文獻(xiàn)使用的PCR方法進(jìn)行鑒定[12]。小鼠脫臼處死后, 外科手術(shù)取出MOE立即置于RNA Later中,按照 TRIzol(Invitrogen)法提取總 RNA, NanoDrop (Thermo)、瓊脂糖凝膠電泳檢測合格后-80℃保存?zhèn)溆?。按照SMARTer PCR cDNA Synthesis Kit(Clontech)反轉(zhuǎn)錄成 cDNA。實驗動物的所有處理方法和操作程序均符合中華人民共和國科學(xué)技術(shù)部頒發(fā)的《關(guān)于善待實驗動物的指導(dǎo)性意見》, 并得到河北大學(xué)動物倫理及關(guān)愛委員會的批準(zhǔn)。

1.2 抑制性消減雜交與文庫構(gòu)建

以來源于 AC3-/-小鼠的 cDNA 作為測試子(Tester)、AC3+/+的cDNA 作為驅(qū)動子(Driver), 進(jìn)行正向消減雜交; AC3+/+小鼠cDNA作為測試子、AC3-/-的cDNA作為驅(qū)動子, 進(jìn)行反向消減雜交。參照PCR Select cDNA Substraction Kit(Clontech)說明書, cDNA用 RsaⅠ酶切后, 測試子分別連接兩種接頭,與過量驅(qū)動子進(jìn)行兩次雜交, 雜交產(chǎn)物采用巢式PCR擴增。每一過程以及最后的消減雜交效率均進(jìn)行檢測驗證。將正向、反向消減雜交第二次PCR擴增產(chǎn)物各3 μL與pESAY-T1(Transgen Biotech, 北京)連接, 轉(zhuǎn)化到大腸桿菌DH5α, 挑選陽性克隆并接種到含有氨芐青霉素(Amp+)LB液體培養(yǎng)基的96孔微孔板內(nèi)進(jìn)行擴大培養(yǎng)。

1.3 斑點雜交(Dot blot)

將擴大培養(yǎng)的陽性克隆菌液進(jìn)行PCR擴增, 分別取 1 μL PCR產(chǎn)物點到兩張帶正電荷的尼龍膜(Amersham, 瑞典)的同一位置, 紫外交聯(lián)(UVP, 美國)固定。以 SSH實驗過程中未經(jīng)過消減雜交的AC3-/-和AC3+/+cDNA樣品為模板, 隨機引物法合成地高辛(DIG)標(biāo)記的正向探針和反向探針(Roche, 瑞士), 按照DIG Labeling and Detection kit(Roche, 瑞士)說明書進(jìn)行Dot blot雜交, NBT/BCIP顯色后用掃描儀(惠普)掃描成像。對比同一克隆在正向、反向探針雜交后顯色程度的不同, 篩選出差異表達(dá)克隆。DNA測序由華大基因科技有限公司(北京)完成。獲得的序列在 NCBI上進(jìn)行 BLASTX比對后, 用AmiGO和 IntroPro網(wǎng)站進(jìn)行蛋白功能注釋, 并在WeGO網(wǎng)站進(jìn)行統(tǒng)計分析。

1.4 qRT-PCR

根據(jù)BLAST分析結(jié)果, 選擇5個差異表達(dá)基因kcnk3、c-mip、mlycd、tmem88b和 trappc5, 進(jìn)行qRT-PCR, 檢測它們在不同個體 AC3-/-(n=3)和AC3+/+小鼠(n=3)中的相對表達(dá)量(引物序列見表1)。

以小鼠MOE組織反轉(zhuǎn)錄(TaKaRa, 日本)后的單鏈cDNA為模板, 使用SYBR熒光染料, 高效PCR酶(Transgen Biotech, 北京)進(jìn)行 qRT-PCR擴增, 用β-actin為內(nèi)參。利用 2-ΔΔct分析方法[13]并結(jié)合SPSS19.0軟件對qRT-PCR擴增結(jié)果進(jìn)行統(tǒng)計分析。

表1 差異表達(dá)及內(nèi)參基因引物序列

2 結(jié)果與分析

2.1 消減雜交效率分析及差異表達(dá)基因片段的富集

為了篩選MOE內(nèi)受AC3調(diào)控的差異表達(dá)基因,以AC3-/-和AC3+/+小鼠MOE mRNA反轉(zhuǎn)錄的cDNA互為測試子和驅(qū)動子, 進(jìn)行雙向消減雜交。從圖1A可以看出, 帶接頭的內(nèi)參基因量(A1、A2)是全部內(nèi)參基因量(G1和G2)的1/4以上, 說明cDNA已與不同的接頭高效連接, 證明了消減雜交的有效性。對照組第二次PCR產(chǎn)物電泳顯示, 摻入的目的片段得到了有效富集(圖 1D), 經(jīng)過消減雜交后的樣品摻入片段清晰顯示在電泳圖中(S-C), 與未經(jīng)過消減雜交樣品相比有更少的彌散條帶和更清晰的目的條帶。同樣, 正向、反向消減雜交后第二次PCR產(chǎn)物電泳顯示出較多特異條帶、較少的彌散(圖1：B, C), 說明差異表達(dá)基因的 cDNA片段得到有效富集。以GAPDH為參照基因?qū)ο麥p雜交效率進(jìn)行PCR法檢測。結(jié)果表明：正向消減組GAPDH基因PCR產(chǎn)物在消減和未消減樣品之間有15個循環(huán)數(shù)的差異, 而反向消減組GAPDH基因PCR產(chǎn)物有10個循環(huán)數(shù)的差異(圖2)。無論是正向還是反向消減雜交, 常量表達(dá)的 GAPDH基因均被大幅度扣除, 說明通過消減雜交非差異表達(dá)基因得到了有效扣除。

2.2 消減文庫的構(gòu)建及差異表達(dá)克隆的篩選

將消減雜交富集的差異表達(dá)基因片段與T載體連接, 分別構(gòu)建正向和反向消減雜交文庫, 共獲得3 958個陽性克隆, 將其在96孔板中擴大培養(yǎng)。每96孔板隨機選取8個陽性克隆用M13F和M13R引物進(jìn)行PCR擴增, 電泳結(jié)果顯示這8個克隆均能擴增100 bp以上的目的片段(電泳圖未給出), 說明挑選的陽性克隆高效攜帶目的片段。將消減雜交文庫用DIG標(biāo)記的正向和反向未消減cDNA探針(濃度均為1000 ng/mL)進(jìn)行Dot blot雜交, NBT/BCIP顯色, 根據(jù)雜交后斑點的大小及顯色深淺(圖3), 初步篩選出386個克隆在AC3-/-和AC3+/+基因型小鼠之間的差異表達(dá), 其中172個為上調(diào)表達(dá), 214個為下調(diào)表達(dá)。

2.3 差異表達(dá)克隆測序及生物信息學(xué)分析

從 386個差異表達(dá)克隆中隨機選取 80個進(jìn)行DNA測序, 序列經(jīng)NCBI數(shù)據(jù)庫比對后獲取相應(yīng)的基因信息。結(jié)果顯示24個上調(diào)克隆中有6個對應(yīng)于kcnk3基因, 3個分別對應(yīng)于mapk7、megf11基因; 38個下調(diào)克隆中有8個對應(yīng)于tmem88b基因, 4個分別對應(yīng)于c-mip基因和skp1a, 3個對應(yīng)于mlycd基因(表2, 表 3), 其余基因相對應(yīng)的差異表達(dá)克隆數(shù)為 1～2個。為了進(jìn)一步分析這些差異表達(dá)基因的功能, 將這些基因序列上傳到AmiGO和IntroPro網(wǎng)站進(jìn)行蛋白功能注釋, 返回數(shù)據(jù)匯總后在 WeGo網(wǎng)站進(jìn)行統(tǒng)計分析。發(fā)現(xiàn)這些差異表達(dá)基因的功能主要集中在分子結(jié)合、細(xì)胞周期、生物和細(xì)胞過程等方面, 上調(diào)差異表達(dá)基因以分子結(jié)合功能最多, 下調(diào)表達(dá)差異基因以細(xì)胞過程最多(圖4)。

圖1 抑制性消減雜交過程驗證結(jié)果A:接頭連接效率檢測。A1(Adaptor1, 接頭1)與G1(GAPDH-A1組)檢測A1與RsaⅠ酶切后cDNA片段的連接效率, A2(Adaptor2R, 接頭2R)與G2(GAPDH-A2組)檢測接頭2R與RsaⅠ酶切后cDNA片段的連接效率; 圖中顯示連接效率均大于1/4。B、C、D：正向、反向和對照組消減后(Substracted, S)與未消減(Unsubstracted, US)樣品第二次PCR電泳結(jié)果, 消減后(S-1、S-2、S-C)比未消減樣品(US-1、US-2、US-C)均有較少彌散和較多擴增條帶, 表明正向和反向消減雜交效果明顯。

圖2 正反向消減雜交效率檢測結(jié)果利用內(nèi)參基因 GAPDH對正向、反向消減雜交效率進(jìn)行檢測, GAPDH基因在正向消減雜交效率檢測中有15個循環(huán)的差異, 在反向消減雜交效率檢測中有10個循環(huán)的差異, 表明消減雜交成功。

圖3 正向和反向DIG標(biāo)記探針斑點雜交結(jié)果A：正向未消減的 US-1探針雜交結(jié)果; B：反向未消減的 US-2探針雜交結(jié)果。通過比較相同位置斑點雜交結(jié)果鑒別差異表達(dá)克隆(箭頭所示)。

表2 上調(diào)差異表達(dá)基因BLAST結(jié)果

表3 下調(diào)差異表達(dá)基因BLAST結(jié)果

圖4 差異表達(dá)基因功能注釋結(jié)果A：上調(diào)差異表達(dá)基因功能注釋后WeGO統(tǒng)計結(jié)果; B：下調(diào)差異表達(dá)基因功能注釋后WeGO統(tǒng)計結(jié)果。

3.4 差異表達(dá)基因相對表達(dá)量分析

為了進(jìn)一步檢測篩選出的受AC3調(diào)控的基因在AC3-/-和AC3+/+小鼠MOE中相對表達(dá)量是否存在差異, 隨機選取kcnk3、c-mip、mlycd、tmem88b、trappc5基因進(jìn)行qRT-PCR分析。從圖5中可以看出, kcnk3基因qRT-PCR結(jié)果呈明顯上升趨勢, 相對表達(dá)量為野生型小鼠的 1.27倍, C-mip、mlycd、tmem88b和trappc5基因qRT-PCR結(jié)果則呈明顯下調(diào)趨勢, 相對表達(dá)量分別為野生型小鼠的20%、7%、32%和29%,而且差異顯著(P＜0.05)。這些結(jié)果表明, 選取的5個基因qRT-PCR檢測的相對表達(dá)量分別與斑點雜交篩選結(jié)果相一致。

圖5 差異表達(dá)基因qRT-PCR結(jié)果以 AC3+/+小鼠(n=3)為對照, AC3-/-小鼠(n=3)MOE內(nèi)各個基因相對表達(dá)量, *表示顯著差異(P＜0.05)。

3 討 論

AC3基因在小鼠的嗅覺活動中起著重要作用,它的缺失不僅引起小鼠嗅覺探測喪失及相關(guān)行為失常, 還會導(dǎo)致MOE內(nèi)cAMP和IP3信號通路異常[4]。然而小鼠 MOE中該基因缺失后是否影響相關(guān)基因網(wǎng)絡(luò)群體的表達(dá), 尚待確定。本文目的是利用 SSH方法和基因敲除小鼠篩選與鑒定MOE內(nèi)受AC3調(diào)控的相關(guān)差異表達(dá)基因。

SSH方法是建立在抑制性雜交和選擇性PCR基礎(chǔ)上的差異表達(dá)基因篩選手段[14], 由于其高效和低假陽性等特點, 廣泛應(yīng)用在動物[15,16]、植物[17]、人類[18]及癌癥[19]等研究領(lǐng)域。Tan等[20]在高血糖發(fā)病研究中, 利用 SSH 方法發(fā)現(xiàn)丙酮酸羧激酶(Phosphoenolpyruvatecarboxykinase, PEPCK)和膽固醇O-乙?；D(zhuǎn)移酶(Sterol O-acyltransferase, SOAT2)兩個基因與葡萄糖激酶(Glucokinase, GCK)代謝緊密相關(guān), 并利用 qRT-PCR對其進(jìn)行了驗證; 在進(jìn)行GCK對腎臟功能影響研究時篩選到谷胱甘肽過氧化物酶 3(Glutathione peroxidase-3, GPX3)基因表達(dá)發(fā)生改變, 并且 GPX3可作為腎臟損傷初期的標(biāo)記物[21]。Wang等[22]利用 SSH 方法鑒定出旋毛蟲(T. spiralis)肌肉期幼蟲與腸道期幼蟲的9個差異表達(dá)基因(Ts7、Ts8、Ts11、Ts17、Ts19、Ts22、Ts23、Ts26、Ts33), 對旋毛蟲肌肉期幼蟲轉(zhuǎn)變成腸道期幼蟲的相關(guān)機理進(jìn)行了闡述。本文利用SSH方法對小鼠MOE內(nèi)受AC3調(diào)控的差異表達(dá)基因進(jìn)行了篩選, 獲得了kcnk3、mapk7、megf11、c-mip、skp1a、mlycd、tmem88b、trappc5、ppat、ssr3等差異表達(dá)基因(表 2, 表 3)。這些基因分別與K+通道[23]、胞外信號調(diào)控[24]、胞內(nèi)蛋白泛素化因子[25]、胞內(nèi)蛋白轉(zhuǎn)膜與運輸[26,27]等生物學(xué)功能相關(guān)。本研究對其中的kcnk3、c-mip、mlycd、 tmem88b和trappc5基因進(jìn)行了qRT-PCR驗證, 證實kcnk3基因在AC3-/-小鼠MOE(相對于野生型小鼠)顯著上調(diào)表達(dá), 而其余4個基因則顯著下調(diào)表達(dá)。

kcnk3為 K+通道的重要成分[28], 它在神經(jīng)系統(tǒng)中起著重要作用[29]。有研究表明, 腺苷酸環(huán)化酶激活肽對 Kv1.4和 Kv4.2有抑制作用[30], 而本文檢測到kcnk3呈上調(diào)表達(dá), 推測AC3可能對K+通道具有負(fù)向調(diào)節(jié)作用, 或者cAMP信號通路與K+通道之間存在交叉對話。另有研究顯示, kcnk3在蛋白激酶C(PKC)的介導(dǎo)下參與內(nèi)吞過程[19], 在細(xì)胞與外界交流中發(fā)揮作用。C-mip通過 c-maf參與 IL-2R及JAK/STAT信號通路的調(diào)控[31,32], 在細(xì)胞的發(fā)育與分化中起著重要作用。此外, C-mip的上調(diào)表達(dá)還與膜性腎病密切相關(guān)[33], 進(jìn)而影響腎臟小球發(fā)育。C-mip也能下調(diào) NF-κB活性, 促進(jìn)足細(xì)胞的凋亡[34]。C-mip還與 p85協(xié)同參與免疫系統(tǒng)發(fā)育過程[35]。AC3基因是否通過c-mip影響MOE內(nèi)嗅覺神經(jīng)細(xì)胞的發(fā)育與分化, 尚待確定。本文檢測到與脂肪酸代謝相關(guān)的 mlycd(MCD)基因在 AC3-/-小鼠MOE內(nèi)呈下調(diào)表達(dá)。MCD是乙酰輔酶A形成過程中的有效抑制物, MCD表達(dá)量降低會使乙酰輔酶A在細(xì)胞內(nèi)含量發(fā)生變化[36～38], 最終影響 MOE內(nèi)脂肪酸及能量代謝。MCD缺失患者纖維母細(xì)胞線粒體脂肪酸代謝異常[39], 影響脂肪酸的生物合成。在缺失AC3基因的小鼠MOE內(nèi), MCD如何對嗅覺或者嗅覺神經(jīng)細(xì)胞發(fā)育和代謝等產(chǎn)生影響, 尚待明確。轉(zhuǎn)膜蛋白基因tmem88b和trappc5被認(rèn)為與胞內(nèi)蛋白轉(zhuǎn)膜和運輸相關(guān)[27,40], 本文中它們的表達(dá)量在AC3-/-小鼠 MOE 內(nèi)顯著下調(diào)。Tmem88 是Wnt/β-catenin信號通路中的散亂(Dishevelled- binding protein)結(jié)合蛋白, RNAi干擾 tmem88能激活Wnt/β-catenin信號。Tmem88作為心肌細(xì)胞 GATA因子的下游調(diào)控元素調(diào)節(jié)心肌細(xì)胞的發(fā)育[41], 還與非小細(xì)胞肺癌調(diào)控有關(guān)[42]。Trappc5與其他因子形成蛋白轉(zhuǎn)運顆粒參與蛋白質(zhì)胞內(nèi)轉(zhuǎn) 運[43]。小鼠 MOE內(nèi)的 1000多個嗅覺受體均為跨膜蛋白, AC3-/-小鼠MOE內(nèi)tmem88b和trappc5表達(dá)量顯著降低, 有可能影響嗅覺受體蛋白的合成或運輸, 進(jìn)而導(dǎo)致嗅覺探測及相關(guān)行為缺陷。嗅覺受體蛋白質(zhì)合成也與能量代謝密切相關(guān), 本研究發(fā)現(xiàn) MCD基因表達(dá)量在AC3-/-小鼠 MOE內(nèi)明顯下調(diào), 這可能是嗅覺受體蛋白合成或分泌受到能量代謝與轉(zhuǎn)運的影響, 以及多個離子通道失調(diào)等綜合原因, 導(dǎo)致 AC3-/-小鼠嗅覺功能喪失[4]。

雖然AC3基因缺失對小鼠MOE產(chǎn)生多方面的影響, 但是 MOE內(nèi)參與其他信號通路(cGMP[44]、IP3[45])的相關(guān)基因在本文中并未檢測出。究其原因：(1)AC3+/+和 AC3-/-基因型小鼠之間僅有一個基因的差異, 它們之間遺傳背景差異較小; (2)與鳥類等研究中篩選出16 512個差異表達(dá)克隆相比較[15], 本文在386個差異表達(dá)克隆中隨機挑取80個進(jìn)行測序分析, 規(guī)模較小, 有可能 cGMP和 IP3信號通路的相關(guān)基因未包括在內(nèi); (3)SSH實驗材料是通過PCR擴增獲得的, 整個過程包括多次酚：氯仿抽提, 這些過程可能會使部分基因丟失。

嗅覺活動對于動物的生存和我們?nèi)祟惖慕】稻哂兄陵P(guān)重要的作用, 但關(guān)于其嗅覺神經(jīng)信號傳導(dǎo)分子機制的了解, 還十分有限。本研究以基因敲除小鼠為材料, 利用 SSH方法成功構(gòu)建了 AC3-/-小鼠MOE組織正向和反向兩個消減文庫, 并對文庫進(jìn)行了篩選、分析與鑒定。發(fā)現(xiàn)所篩選出的差異表達(dá)基因與K+通道、細(xì)胞發(fā)育與分化、脂肪酸代謝和胞內(nèi)蛋白轉(zhuǎn)運等重要生物學(xué)過程相關(guān)。可能是這些基因共同作用結(jié)果, 導(dǎo)致AC3敲除后小鼠嗅覺及其相關(guān)行為表現(xiàn)功能缺陷。本研究結(jié)果為闡明AC3對小鼠嗅覺功能及相關(guān)行為的分子調(diào)控機制提供了一定的數(shù)據(jù)積累。

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(責(zé)任編委：史慶華)

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 Zhou1,2, Zhe Zhang3, Zhihua Ni1, Yuanxiang Hu3, Weili Liu3, Yongchao Li4, Daniel R. Storm5, Runlin Z. Ma4, Zhenshan Wang1,2

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

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.

adenylate cyclase 3 (AC3); main olfactory epithelium (MOE); differentially expressed genes; suppression subtractive hybridization (SSH)

2013-11-24;

2014-01-16

國家自然科學(xué)基金項目(編號：31171191), 河北省自然科學(xué)基金項目(編號：C2012201106), 教育部留學(xué)人員回國啟動基金項目(教外司留＜2013＞693號)和河北省生物工程重點學(xué)科經(jīng)費資助

曹振龍, 碩士研究生, 專業(yè)方向：細(xì)胞分子生物學(xué)。E-mail: dafeizizhu@163.com

王振山, 教授, 博士生導(dǎo)師, 研究方向：動物行為遺傳分子機制。E-mail: zswang@hbu.edu.cn

10.3724/SP.J.1005.2014.0574

時間: 2014-4-1 11:35:35

URL: http://www.cnki.net/kcms/detail/11.1913.R.20140401.1135.001.html