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豬ESRRB啟動(dòng)子克隆及其調(diào)控活性檢測(cè)

2015-07-19 13:05:10楊藩王亞嫻杜麗霞王華巖
生物工程學(xué)報(bào) 2015年4期
關(guān)鍵詞:克隆干細(xì)胞試劑盒

楊藩,王亞嫻,杜麗霞,王華巖

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豬啟動(dòng)子克隆及其調(diào)控活性檢測(cè)

楊藩1,王亞嫻1,杜麗霞2,王華巖1

1 西北農(nóng)林科技大學(xué)動(dòng)物醫(yī)學(xué)院陜西省干細(xì)胞工程技術(shù)研究中心,陜西 楊凌 712100 2 西北農(nóng)林科技大學(xué)創(chuàng)新實(shí)驗(yàn)學(xué)院,陜西楊凌 712100

楊藩, 王亞嫻, 杜麗霞, 等. 豬ESRRB啟動(dòng)子克隆及其調(diào)控活性檢測(cè). 生物工程學(xué)報(bào), 2015, 31(4): 491–500.Yang F, Wang YX, Du LX, et al. Cloning and regulation of pig estrogen related receptor β gene (ESRRB) promoter. Chin J Biotech, 2015, 31(4): 491–500.

(Estrogen related receptor β) 屬于雌激素受體家族,是一類在胚胎早期外胚層細(xì)胞中表達(dá)并對(duì)干細(xì)胞多能性維持起重要作用的基因。為了探索豬的表達(dá)和轉(zhuǎn)錄調(diào)控機(jī)制,克隆了3.3 kb啟動(dòng)子片段,構(gòu)建了相應(yīng)的報(bào)告載體。并將報(bào)告載體分別轉(zhuǎn)染293T人胚腎細(xì)胞、Hela人宮頸癌細(xì)胞和小鼠C2C12成肌細(xì)胞。通過(guò)TFSEARCH和JASPER方法對(duì)啟動(dòng)子潛在的轉(zhuǎn)錄調(diào)控位點(diǎn)進(jìn)行分析,發(fā)現(xiàn)該啟動(dòng)子上有SMAD、STAT3、MYC、KLF4等多能轉(zhuǎn)錄因子的結(jié)合位點(diǎn)。將相應(yīng)的轉(zhuǎn)錄因子與啟動(dòng)子共轉(zhuǎn)染,并檢測(cè)報(bào)告基因熒光素酶的活性。結(jié)果顯示豬B啟動(dòng)子具有明顯的組織特異性調(diào)控,同時(shí)SMAD對(duì)啟動(dòng)子活性有較明顯的調(diào)控作用。進(jìn)一步對(duì)3.3 kb片段進(jìn)行了一系列的缺失,發(fā)現(xiàn)豬核心區(qū)域位于5′上游的?25 bp和?269 bp之間。研究結(jié)果表明豬啟動(dòng)子上潛在的轉(zhuǎn)錄因子結(jié)合位點(diǎn)及啟動(dòng)子核心區(qū)域是參與調(diào)控表達(dá)的重要序列。

豬,雌激素相關(guān)受體,啟動(dòng)子,多能干細(xì)胞,轉(zhuǎn)錄調(diào)控

多能干細(xì)胞的自我更新需要相關(guān)信號(hào)通路和各種轉(zhuǎn)錄因子調(diào)控來(lái)維持,其中ERK、GSK、LIF等通路和Oct4、Sox2、Nanog等因子,在其中起著關(guān)鍵作用[1]。目前,小鼠胚胎干細(xì)胞 (ESCs) 可以在添加2i/LiF的培養(yǎng)基中穩(wěn)定傳代[2],其中2i通過(guò)抑制ERK和GSK3通路發(fā)揮維持ESCs自我更新和多能性的作用。GSK3通過(guò)β-catein對(duì)許多基因起著負(fù)調(diào)控的作用,這其中就包括c-Myc[3-4]。同時(shí),遺傳分析表明β-catein通過(guò)Tcf3協(xié)同作用負(fù)調(diào)控ESCs的自我更新[5]。為了明確Tcf3的下游靶基因,Simth研究團(tuán)隊(duì)敲除了干細(xì)胞的基因,并結(jié)合Tcf3的調(diào)控位點(diǎn)發(fā)現(xiàn)雌激素相關(guān)受體b(Esrrb)是Tcf3的下游靶基因,對(duì)干細(xì)胞自我更新的維持起著重要的作用[6]。Chambers研究團(tuán)隊(duì)在分析Nanog的下游靶基因時(shí)發(fā)現(xiàn),Nanog可以激活Esrrb的表達(dá)并維持干細(xì)胞的自我更新[7]。同時(shí),Esrrb或者Nanog的過(guò)表達(dá)可以在不添加LIF的條件下維持干細(xì)胞的自我更新。

雌激素相關(guān)受體(Estrogen related receptor) 屬于核受體家族。目前己發(fā)現(xiàn)3種亞型,即[8]、[8]、[9]。雌激素受體基因包含有相對(duì)保守的結(jié)構(gòu)區(qū)域:配體結(jié)合域 (LBD) 和DNA結(jié)合域(DBD)[8,10]。該家族基因可以不經(jīng)過(guò)相應(yīng)激素配體的刺激而直接入核并調(diào)控基因的表達(dá)。相對(duì)于和,的時(shí)空表達(dá)特異性較高。在小鼠胚胎發(fā)育到5.5 d時(shí)出現(xiàn),并隨著胚胎的進(jìn)一步發(fā)育而逐漸消失,其缺失會(huì)導(dǎo)致胚胎發(fā)育不正常[11-12]。因此,開(kāi)展對(duì)該基因啟動(dòng)子的研究,有助于揭示多能干細(xì)胞的自我更新維持和多向分化能力的調(diào)控機(jī)制。本研究為揭示豬基因在分子調(diào)控機(jī)理,分子克隆了啟動(dòng)子片段,并對(duì)啟動(dòng)子上的調(diào)控位點(diǎn)進(jìn)行了系統(tǒng)分析,通過(guò)對(duì)豬啟動(dòng)子缺失,用雙熒光素酶表達(dá)載體驗(yàn)證了潛在轉(zhuǎn)錄因子對(duì)的調(diào)控。本研究為進(jìn)一步探索對(duì)豬多能干細(xì)胞自我更新的維持和調(diào)控機(jī)理奠定了基礎(chǔ)。

1 材料與方法

1.1 菌種、載體和細(xì)胞

感受態(tài)大腸桿菌DH5α、質(zhì)粒pEGFP-1、pEGFP-C1、pGL3-Basic、pMX-OCT4、pMX-CMYC、pCDNA-LIF、pEGFPC1-SMAD2、pEGFPC1- SMAD3、pEGFPC1-SMAD7、人胚腎細(xì)胞(293T)、人宮頸癌細(xì)胞(Hela)、小鼠成肌細(xì)胞(C2C12) 和豬胎兒成纖維細(xì)胞(PEF) 由陜西省干細(xì)胞工程技術(shù)研究中心保存。載體pGEM-T Easy及熒光素酶檢測(cè)試劑盒Dual-Luciferase?Reporter Assay System均購(gòu)自Promega公司。豬各組織器官采集于陜西萬(wàn)盛肉類加工有限公司的屠宰場(chǎng)。

1.2 試劑及耗材

總RNA提取試劑盒、基因組提取試劑盒、質(zhì)粒提取試劑盒、凝膠回收試劑盒、DNA marker均購(gòu)自天根公司;限制性內(nèi)切酶、T4 DNA連接酶、反轉(zhuǎn)錄試劑盒和聚合酶均購(gòu)自Fermentas公司;高保真DNA聚合(PhantaTMHS Super-Fidelity DNA Polymerase) 購(gòu)自Vazyme公司;定量PCR Mix (Power 2xSYBR Real-time PCR Premixture) 購(gòu)自于Bioteke公司;脂質(zhì)體 (Lipofectamine 2 000)、Opti-MEM和DMEM培養(yǎng)基均購(gòu)自Invitrogen公司;非必需氨基酸 (NEAA)、L-谷氨酰胺 (L-Glu)、β-巰基乙醇 (β-ME) 購(gòu)自Gibco公司;胎牛血清(FBS) 購(gòu)自Hyclone公司;PCR 引物由華大基因科技服務(wù)有限公司合成。

1.3啟動(dòng)子的克隆與載體構(gòu)建

參照血液/細(xì)胞/組織基因組DNA提取試劑盒 (天根公司),從PEF細(xì)胞中提取豬基因組DNA。從UCSC (http://genome.ucsc.edu/) 網(wǎng)站下載豬的5¢上游序列,設(shè)計(jì)帶有酶切位點(diǎn)d Ⅲ和Ⅰ的啟動(dòng)子上下游引物,并擴(kuò)增3.3 kb啟動(dòng)子片段。產(chǎn)物用瓊脂糖凝膠DNA回收試劑盒進(jìn)行回收,并與pGEM-T Easy載體連接,連接產(chǎn)物轉(zhuǎn)化到大腸桿菌DH5α中。挑取陽(yáng)性克隆提取質(zhì)粒,經(jīng)限制性內(nèi)切酶Ⅰ、dⅢ雙酶切鑒定。酶切鑒定正確的質(zhì)粒送華大生物工程有限公司測(cè)序。測(cè)序正確的質(zhì)粒通過(guò)dⅢ和Ⅰ與pEGFP-1和pGL3-Basic連接,并分別命名為pE3.3和pL3.3。以pE3.3為模板,分別設(shè)計(jì)帶有酶切位點(diǎn)Ⅰ和dⅢ的上下游引物,擴(kuò)增2 093 bp、1 503 bp、876 bp、575 bp和308 bp的啟動(dòng)子片段,亞克隆到pGL3-Basic報(bào)告載體中,分別命名為pL2.0、pL1.5、pL0.8、pL0.5和pL0.3。引物序列見(jiàn)表1。

1.4 細(xì)胞培養(yǎng)與轉(zhuǎn)染

293T、Hela和C2C12細(xì)胞用含有10%胎牛血清的DMEM培養(yǎng)液,在37 ℃、含有5% CO2的細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng)。按照Lipofectine 2 000使用說(shuō)明書,首先將pE3.3啟動(dòng)子分別轉(zhuǎn)入293T、Hela和C2C12細(xì)胞,轉(zhuǎn)染后48 h在熒光顯微鏡下觀察綠色熒光。同時(shí),將293T細(xì)胞按2.4×104/孔接種于48孔培養(yǎng)板中,待細(xì)胞完全貼壁覆蓋率至50%?60%時(shí),將pL3.3、pL2.0、pL1.5、pL0.8、pL0.5和pL0.3等啟動(dòng)子熒光報(bào)告載體 (500 ng) 和pCMV-Renilla表達(dá)載體 (20 ng) 共同轉(zhuǎn)入細(xì)胞,48 h后收集細(xì)胞并進(jìn)行熒光素酶活性檢測(cè)。每個(gè)啟動(dòng)子做3次獨(dú)立實(shí)驗(yàn),每次3個(gè)重復(fù)。

表1 引物序列表

1.5 熒光定量PCR

按照總RNA提取試劑盒說(shuō)明書 (天根公司)提取組織總RNA,按照RevertAidTMFrist Strand cDNA Synthesis Kit相關(guān)說(shuō)明進(jìn)行反轉(zhuǎn)錄獲得cDNA,并設(shè)計(jì)qESRRB和qGAPDH引物。熒光定量PCR反應(yīng)體系如下:2×SYBR Green Mix 10 μL,25 mmol/L dNTPs 1 μL,上、下游引物各0.5 μL (10 μmol/L),模板cDNA 1 μL,去離子水7 μL,共20 μL。每個(gè)樣本設(shè)3個(gè)重復(fù)。反應(yīng)條件為:95 ℃ 30 s;95 ℃ 5 s,60 ℃ 30 s,共40個(gè)循環(huán)。實(shí)時(shí)熒光定量PCR引物序列見(jiàn)表1。

1.6 統(tǒng)計(jì)學(xué)分析

所有數(shù)據(jù)均用平均值±標(biāo)準(zhǔn)偏差表示,并經(jīng)雙尾-test檢驗(yàn)差異是否具有統(tǒng)計(jì)學(xué)意義。*表<0.05,**表示<0.01。

2 結(jié)果與分析

2.1 豬啟動(dòng)子克隆及生物學(xué)分析

本研究從豬的基因組中克隆獲得了3 307 bp的基因5¢端上游片段(圖1A)。用生物信息學(xué)的方法發(fā)現(xiàn)序列上存在很多與多能性轉(zhuǎn)錄因子結(jié)合的位點(diǎn),如:KLF4、MYC、SMAD和STAT3等(圖1B)。但是在轉(zhuǎn)錄起始位點(diǎn)上游缺乏經(jīng)典的TATA-box (TATAAA)。

圖1 ESRRB 3 kb啟動(dòng)子克隆及生物信息學(xué)分析

2.2 多能性相關(guān)基因?qū)φ{(diào)控分析

通過(guò)在線啟動(dòng)子分析軟件JASPAR (http://jaspar.genereg.net/) 分析豬啟動(dòng)子,發(fā)現(xiàn)KLF4 (–2 582,–1 488,–1 059,–860,–591,–123);MYC (–1 759,–1 288);STAT3 (–2 944,–2 067,–2 052,–1 316,–801)和SMAD(–2 723,–1 924,–1 414,–599) 等的可能結(jié)合位點(diǎn)。但是,未發(fā)現(xiàn)OCT4和SOX2的結(jié)合位點(diǎn)(圖2A)。通過(guò)將構(gòu)建好的pL3.3分別與pMX-CMYC、pCDNA-LIF、pEGFPC1-SMAD2、pEGFPC1-SMAD3和pEGFPC1-SMAD2+ pEGFPC1-SMAD3共轉(zhuǎn)染293T細(xì)胞,發(fā)現(xiàn)這些因子都能一定程度地激活啟動(dòng)子,其中SMAD2 + SMAD3的作用最為明顯,是只加入pL3.3的對(duì)照組的2.7倍(圖2B)。因?yàn)闆](méi)有SOX2的結(jié)合位點(diǎn),pEGFP-SOX2并未提高的表達(dá)水平。

圖2 多能性基因?qū)ωiESRRB啟動(dòng)子的調(diào)控

2.3 豬組織特異性表達(dá)檢測(cè)

通過(guò)實(shí)時(shí)定量PCR對(duì)胸腺、脂肪、皮膚、肌肉、心臟、肝臟、腦、睪丸、卵巢、脾臟、腎臟和豬成纖維細(xì)胞中的表達(dá)情況進(jìn)行了測(cè)定。結(jié)果顯示在腎臟表達(dá)最高,在與生殖相關(guān)的組織如睪丸卵巢中也有表達(dá)。而在脂肪、皮膚和肌肉等組織中的表達(dá)較低(圖3A)。這個(gè)結(jié)果與人和鼠的報(bào)道相似[13-14]。對(duì)攜帶3.3 kb啟動(dòng)子片段的pE3.3進(jìn)行d Ⅲ和Ⅰ酶切鑒定,結(jié)果表明插入片段正確(圖3B)。將pE3.3報(bào)告載體分別轉(zhuǎn)入C2C12、Hela和293T細(xì)胞,48 h后在熒光顯微鏡下觀察細(xì)胞表達(dá)GFP情況。結(jié)果顯示,在293T細(xì)胞中有GFP的表達(dá),而在C2C12與Hela細(xì)胞中沒(méi)有GFP的表達(dá)(圖3C)。這一結(jié)果印證了啟動(dòng)子在腎臟細(xì)胞中的高表達(dá)特性 (圖3A)。

圖3 定量檢測(cè)ESRRB的組織特異性表達(dá)

2.4 構(gòu)建啟動(dòng)子片段缺失載體

以重組質(zhì)粒pE3.3為模板用不同的引物對(duì)啟動(dòng)子5?端上游序列進(jìn)行缺失,獲得 2 093 bp、1 503 bp、876 bp、575 bp和308 bp等片段,經(jīng)瓊脂糖凝膠電泳分析驗(yàn)證,PCR擴(kuò)增產(chǎn)物與預(yù)期片段大小相符(圖4A)。分別將這些片段插入到pGL3-basic質(zhì)粒,構(gòu)建pL2.0、pL1.5、pL0.8、pL0.5和pL0.3報(bào)告載體,并經(jīng)限制性內(nèi)切酶d Ⅲ和Ⅰ雙酶切鑒定 (圖4B)。檢測(cè)結(jié)果表明酶切目的片段大小與預(yù)期吻合,載體構(gòu)建正確。

2.5 篩選豬啟動(dòng)子核心調(diào)控區(qū)

將攜帶不同啟動(dòng)子片段的質(zhì)粒pL3.3、pL2.0、pL1.5、pL0.8、pL0.5和pL0.3等,瞬時(shí)轉(zhuǎn)染293T細(xì)胞48 h后,對(duì)報(bào)告基因熒光素酶活性進(jìn)行檢測(cè)。結(jié)果顯示,pL0.3沒(méi)有啟動(dòng)子活性,而pL0.5的啟動(dòng)子活性與對(duì)照組相比提高了約50倍。pL0.5相比于pL0.3多出了–269 bp至–25 bp之間的序列,表明該段序列存在一個(gè)正調(diào)控域是該啟動(dòng)子的核心調(diào)控區(qū)域。進(jìn)一步測(cè)定顯示pL0.8、pL1.5、pL2.0和pL3.3的活性比pL0.5的又提高了近70%,說(shuō)明在啟動(dòng)子–0.5至–0.8 bp之間的序列,還存在一個(gè)正調(diào)控域 (圖5)。這兩個(gè)調(diào)控域的調(diào)控元件和生物學(xué)功能還有待進(jìn)一步探索研究。

圖4 ESRRB啟動(dòng)子的PCR缺失及相應(yīng)的熒光素酶報(bào)告載體構(gòu)建

圖5 豬ESRRB不同長(zhǎng)度啟動(dòng)子的活性檢測(cè)

3 討論

雌激素家族相關(guān)受體基因β是維持ESCs自我更新的重要轉(zhuǎn)錄因子,抑制其表達(dá)會(huì)直接導(dǎo)致ESCs的分化[15]。研究發(fā)現(xiàn)Esrrb能夠與其他多能轉(zhuǎn)錄因子相互作用,在ESCs中與Oct4、Sox2、Nanog和Klf4擁有共同的結(jié)合位點(diǎn)[16];同時(shí),啟動(dòng)子上存在Oct4、Nanog和Tcf3的結(jié)合位點(diǎn)[6]。本研究克隆了豬啟動(dòng)子,經(jīng)生物信息學(xué)分析表明啟動(dòng)子上存在潛在的、、和的結(jié)合位點(diǎn)。通過(guò)雙熒光素酶報(bào)告載體實(shí)驗(yàn)發(fā)現(xiàn),這些轉(zhuǎn)錄因子都能夠一定程度地激活豬啟動(dòng)子活性。其中的效果最為顯著。所介導(dǎo)的信號(hào)通路Nodal/Activn能夠促進(jìn)小鼠胚胎干細(xì)胞的增殖和自我更新[17]。該結(jié)果也表明通過(guò)與不同基因和通路的協(xié)同作用實(shí)現(xiàn)對(duì)自我更新的維持[18]。

通過(guò)實(shí)時(shí)定量PCR,我們發(fā)現(xiàn)主要在腎臟、脾臟、卵巢和睪丸中表達(dá)。將pE3.3分別轉(zhuǎn)入到人胚腎細(xì)胞293T、宮頸癌細(xì)胞Hela和小鼠成肌細(xì)胞C2C12中,表現(xiàn)出組織特異性表達(dá),只在腎臟來(lái)源的293T中有綠色熒光表達(dá)。對(duì)比的組織表達(dá)譜研究表明,人的主要分布在腎臟和睪丸[19];而小鼠的主要分布于腎臟和心臟[20];大鼠的分布要廣泛一些,在腎臟、心臟、睪丸和神經(jīng)內(nèi)分泌相關(guān)的組織中均檢測(cè)到表達(dá)[8]。通過(guò)比較豬、人、小鼠和大鼠的組織表達(dá)譜發(fā)現(xiàn),在物種間表達(dá)有一定的差異,同時(shí)腎臟是表達(dá)的一個(gè)重要器官??赡茉谄渲袇⑴c調(diào)節(jié)腎臟分泌固醇類激素[21]。

有報(bào)道通過(guò)對(duì)豬Ⅱ(UPII)[22]、[23]和[24]等基因的啟動(dòng)子進(jìn)行缺失發(fā)現(xiàn)了相應(yīng)的核心啟動(dòng)子。為了進(jìn)一步分析啟動(dòng)子的核心調(diào)控區(qū)域,我們對(duì)其3.3 kb啟動(dòng)子進(jìn)行了缺失。結(jié)果發(fā)現(xiàn)位于5?端上游–25 bp和–269 bp序列區(qū)有兩個(gè)轉(zhuǎn)錄調(diào)控域,能夠明顯調(diào)控啟動(dòng)子活性。進(jìn)一步分析發(fā)現(xiàn),不存在TATA box,是TATA-less基因。事實(shí)上TATA box作為典型的核心啟動(dòng)子元件并非存在于所有的基因中。相反,大多數(shù)基因在轉(zhuǎn)錄的時(shí)候不需要TATA box[25]。

本文通過(guò)對(duì)豬組織特異表達(dá)的檢測(cè)和對(duì)啟動(dòng)子核心調(diào)控區(qū)的測(cè)定,初步揭示了該基因的表達(dá)和調(diào)控特點(diǎn),為進(jìn)一步確定的調(diào)控機(jī)制及其對(duì)多能性的維持機(jī)理奠定了基礎(chǔ)。同時(shí),構(gòu)建的pE3.3報(bào)告載體也可以用于示蹤細(xì)胞重編程,為開(kāi)展豬誘導(dǎo)多能干細(xì)胞的建系提供了新的手段。

REFERENCES

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(本文責(zé)編郝麗芳)

Cloning and regulation of pig estrogen related receptor β gene () promoter

Fan Yang1, Yaxian Wang1, Lixia Du2, and Huayan Wang1

1 Shaanxi Center for Stem Cell Engineering and Technology, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China 2 Innovation Experimental College, Northwest A&F University, Yangling 712100, Shaanxi, China

The estrogen related receptor family member(Estrogen related receptor β) is a gene that expresses in the early stage of embryo and plays an important role in the core pluripotent network. Its function has been analyzed in human and mouse, although no report so far related to pig. Therefore, to explore its mechanism of transcriptional regulation and expression pattern, we cloned a 3.3 kb pigpromoter by PCR and constructed the green fluorescence protein (GFP) reporter vector pE3.3. We used these vectors to study theexpression pattern in 293T, Hela and C2C12. Sequence was analyzed for regulatory elements that share homology to known transcription factor binding sites by TFSEARCH and JASPER program. Some pluripotency related genes such as SMAD, STAT3, MYC, KLF4 and ESRRB have been found within the 3.3 kb sequence by co-transfected pigpromoter and these potential regulators. We found that ESRRB only expressed in 293T and SMAD could activateexpression obviously. To determine the core promoter region, a series ofpromoter fragments with gradually truncated 5?-end were produced by PCR and inserted into pGL3-Basic vector. After transient transfection into 293T, dual luciferase assay was used to measure these promoter activities. The result suggested that the core promoter of piglocated within ?25 bp to ?269 bp region. These results suggest that these transcription factor binding sites and the core promoter region may be essential for transcriptional regulation of piggene.

pig,, promoter, pluripotent cells, transcriptional regulation

July 14, 2014; Accepted:October 27, 2014

Huayan Wang. Tel: +86-29-87080069; E-mail: hhwang101@163.com

Supported by: National Natural Science Foundation of China (No. 31371505), National Basic Research Program of China (973 Program) (No. 2011CBA01002).

國(guó)家自然科學(xué)基金(No. 31371505), 國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃 (973計(jì)劃) (No. 2011CBA01002) 資助。

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