奶牛SREBP1蛋白在乳腺上皮細(xì)胞的表達(dá)定位及對(duì)SCD1基因啟動(dòng)子的轉(zhuǎn)錄調(diào)控

韓立強(qiáng)，王月影，王林楓，朱河水，鐘 凱，褚貝貝，楊國(guó)宇

（河南農(nóng)業(yè)大學(xué)牧醫(yī)工程學(xué)院/農(nóng)業(yè)部動(dòng)物生化與營(yíng)養(yǎng)重點(diǎn)實(shí)驗(yàn)室，鄭州 450002）

奶牛SREBP1蛋白在乳腺上皮細(xì)胞的表達(dá)定位及對(duì)SCD1基因啟動(dòng)子的轉(zhuǎn)錄調(diào)控

韓立強(qiáng)，王月影，王林楓，朱河水，鐘 凱，褚貝貝，楊國(guó)宇

（河南農(nóng)業(yè)大學(xué)牧醫(yī)工程學(xué)院/農(nóng)業(yè)部動(dòng)物生化與營(yíng)養(yǎng)重點(diǎn)實(shí)驗(yàn)室，鄭州 450002）

【目的】固醇調(diào)節(jié)元件結(jié)合蛋白 1( SREBP1)作為核轉(zhuǎn)錄因子，對(duì)于細(xì)胞脂肪合成酶基因的表達(dá)發(fā)揮著重要的調(diào)控作用。論文旨在奶牛乳腺上皮細(xì)胞中研究SREBP1對(duì)于SCD1基因啟動(dòng)子的轉(zhuǎn)錄調(diào)控作用，為進(jìn)一步明確SREBP1對(duì)于靶基因的轉(zhuǎn)錄調(diào)控機(jī)制提供理論基礎(chǔ)。【方法】以荷斯坦奶牛乳腺組織的cDNA為模板，采用分段克隆的方法獲得SREBP1基因的編碼序列，通過(guò)重組酶與pcDNA3.1載體進(jìn)行重組環(huán)化構(gòu)建pcDNA3.1-SREBP1表達(dá)載體，將構(gòu)建的載體測(cè)序驗(yàn)證后提取質(zhì)粒，轉(zhuǎn)染奶牛乳腺上皮細(xì)胞。以EIF3K基因?yàn)閮?nèi)參基因，采用熒光定量PCR檢測(cè)SREBP1基因mRNA的表達(dá)差異；采用免疫熒光的方法對(duì)SREBP1進(jìn)行標(biāo)記，以DAPI復(fù)染細(xì)胞核，激光共聚焦觀察SREBP1蛋白的亞細(xì)胞定位；轉(zhuǎn)染含有不同調(diào)控元件的SCD基因啟動(dòng)子，同時(shí)轉(zhuǎn)染1.0 μg pcDNA3.1-SREBP1作為處理，熒光素酶報(bào)告基因系統(tǒng)分析啟動(dòng)子活性；分別轉(zhuǎn)染0.25、0.5和1μg的pcDNA3.1-SREBP1載體，分析pGL3-SCD 2 和pGL3-SCD3啟動(dòng)子活性與SREBP1之間的量效關(guān)系?！窘Y(jié)果】分段克隆得到的PCR產(chǎn)物分別為1 170、1 116、363和900 bp的片段，經(jīng)過(guò)與pcDNA3.1載體重組后獲得pcDNA3.1-SREBP1表達(dá)載體，經(jīng)酶切和測(cè)序驗(yàn)證，發(fā)現(xiàn)除1個(gè)無(wú)義突變外，與標(biāo)準(zhǔn)序列完全相同，整個(gè)序列長(zhǎng)度達(dá)到3 510bp；將pcDNA3.1-SREBP1載體轉(zhuǎn)染乳腺上皮細(xì)胞后，Real-time PCR檢測(cè)發(fā)現(xiàn)與轉(zhuǎn)染空載體的對(duì)照組相比，SREBP1基因的mRNA表達(dá)倍數(shù)增強(qiáng)130.4倍（P＜0.001）；激光共聚焦觀察發(fā)現(xiàn)，DAPI染色的細(xì)胞核呈藍(lán)色，免疫熒光標(biāo)記的SREBP1呈綠色，二者融合后呈現(xiàn)青色，共定位在乳腺上皮細(xì)胞核中；啟動(dòng)子活性檢測(cè)發(fā)現(xiàn)，與pGL3-SCD1、pGL3SCD 2相比，SREBP1處理能夠極顯著增加pGL3-SCD3、pGL3-CD4啟動(dòng)子的活性（P＜0.001），分別比對(duì)照組提高了1.0倍和0.7倍，進(jìn)一步分析發(fā)現(xiàn)，在用0.25—1 μg的pcDNA3.1-SREBP1處理后，與pGL3-SCD2的啟動(dòng)子活性持續(xù)下降相比，pGL3-SCD3的啟動(dòng)子活性從59.81上升到108.43（P＜0.001），二者存在劑量效應(yīng)關(guān)系，結(jié)合SCD2和SCD 3啟動(dòng)子上主要的結(jié)構(gòu)差異SRE元件（5′-AGCAGATTGCG-3′），推測(cè)此序列可能是SREBP1調(diào)控SCD基因啟動(dòng)子轉(zhuǎn)錄的結(jié)合序列?！窘Y(jié)論】克隆構(gòu)建奶牛SREBP1基因表達(dá)載體，亞細(xì)胞定位SREBP1蛋白主要在乳腺上皮細(xì)胞核中，SREBP1可以與SRE調(diào)控元件結(jié)合促進(jìn)SCD1基因啟動(dòng)子的轉(zhuǎn)錄。

奶牛；固醇調(diào)節(jié)元件結(jié)合蛋白 1；乳腺上皮細(xì)胞；亞細(xì)胞定位；轉(zhuǎn)錄調(diào)控

Abstract:【Objective】As a member of nuclear transcription factor, sterol regulatory element binding protein1（SREBP1）plays a significant role in the expression of the lipogenic gene . The objective of this study is to investigate the effect of SREBP1 on the regulation of transcription of SCD1 gene in the mammary epithelial cells, which will provide a fundamental basis for the transcription regulation mechanism of SREBP1 to target gene. 【Method】 The coding sequence (CDS) of SREBP1 gene was cloned using the method of subsection cloning with the cDNA of Holstein mammary tissues as the template. The expression vector pcDNA3.1-SREBP1 was recombined and constructed by recombining vector pcDNA3.1. Plasmids were extracted from the constructed vector after being verified by sequencing, and transfected to mammary epithelial cells. With gene EIF3K as the reference gene, the expressional difference of mRNA in gene SREBP1 was detected with Real-time PCR. SREBP1 was marked with the immunofluorescence method. The cell nucleus was restained with DAPI, and the subcellular localization of protein SREBP1 was observed with laser con-focal microscopy. The SCD1 gene promoters with different regulatory elements and 1.0 μg pcDNA3.1-SREBP1 were restained as treatments, and the promoter activity was analyzed systematically with luciferase reporter gene. Vectors pcDNA3.1-SREBP1 of 0.25 μg, 0.5 μg and 1 μg were transfected respectively. The dose-effect relationship between the promoter activity of pGL3-SCD2 , pGL3-SCD3 and protein SREBP1 was analyzed. 【Result】 The PCR products cloned in subsection were fragments 1170bp, 1116bp, 363bp and 900 bp respectively. The expression vector pcDNA3.1-SREBP1 was obtained by reconstructing the fragments and vector pcDNA3.1. Confirmed by digestion and sequence, the expression vector, except one nonsense mutation, was exactly the same with the standard sequence. The whole sequence length reached 3510 bp. After vector pcDNA3.1-SREBP1 was transfected into mammary epithelial cells, compared with the control group of the empty transfected vector, the expression mRNA of gene SREBP1 increased by 130.4 times （P＜0.001）by the real-time PCR detection. As shown in the observation of the laser con-focal microscopy, the cell nucleus dyed with DAPI was blue, and SREBP1 with immunofluorescent labeling was green, both of which were cyan after being merged and were localized in the mammary epithelial cell nucleus. The results of the detection of the promoter activity showed that, compared with those of pGL3-SCD1 and pGL3SCD 2, the treatment with SREBP1 increased the promoter activity of pGL3-SCD3 and pGL3-CD4 significantly （P＜0.001）, and their promoter activity increased by 1.0 and 0.7 times. The further study showed that, after treatment with 0.25-1μg pcDNA3.1-SREBP1, compared with the continuous decrease of the promoter activity of pGL3-SCD2, the promoter activity of pGL3-SCD3 increased from 59.81 to 108.43（P＜0.001）. There is a dose-effect relationship between them. Combined with the element SRE（5′-AGCAGATTGCG-3′）, the architectural difference between promoter SCD2 and promoter SCD 3, it could be speculated that the sequence was the incorporating sequence in which SREBP1 regulated the transcription of gene promoter SCD1.【Conclusion】 It was verified that the gene expression vector SREBP1 was constructed by cloning, the subcellular localization of protein SREBP1 was in the mammary epithelial cell nucleus, and SREBP1 promoted the regulation of the transcription of SCD1 promoter together with regulatory element SRE.

0 引言

【研究意義】乳腺做為牛、羊等哺乳動(dòng)物合成分泌乳汁的主要器官，其基因表達(dá)調(diào)控機(jī)制一直是畜牧工作者進(jìn)行科學(xué)研究的重要內(nèi)容。BIONAZ等采集奶牛乳腺組織分析基因表達(dá)譜[1]，發(fā)現(xiàn)固醇調(diào)節(jié)元件結(jié)合蛋白1（Sterol-regulatory element binding protein1，SREBP1）作為一種轉(zhuǎn)錄因子，是乳腺中脂肪合成代謝基因表達(dá)調(diào)控網(wǎng)絡(luò)的中心，其他研究發(fā)現(xiàn) 一些營(yíng)養(yǎng)因素可以影響 SREBP1蛋白的活性和脂肪合成酶基因的表達(dá)[2-3]。因此研究SREBP1及脂肪合成酶基因在乳腺上皮細(xì)胞中的表達(dá)調(diào)控機(jī)制，可以為闡明營(yíng)養(yǎng)元素影響乳腺脂肪合成的機(jī)制提供理論基礎(chǔ)。【前人研究進(jìn)展】早期在生產(chǎn)中發(fā)現(xiàn)對(duì)奶牛飼喂油脂會(huì)造成奶牛的乳脂率下降，形成生產(chǎn)上低脂乳癥（milk fat depression, MFD）的現(xiàn)象[4]，后來(lái)發(fā)現(xiàn)奶牛在發(fā)生MFD時(shí)，不僅乳脂肪含量與組成發(fā)生變化，而且乳腺組織的脂肪合成關(guān)鍵酶的基因表達(dá)也受到抑制[4-6]，進(jìn)一步研究發(fā)現(xiàn)在降低脂肪合成酶基因表達(dá)的同時(shí)也能夠影響SREBP1基因mRNA的表達(dá)[3，7]。SREBP1作為重要的核轉(zhuǎn)錄因子，主要調(diào)控細(xì)胞內(nèi)的膽固醇濃度和脂類的穩(wěn)態(tài)[8-10]。對(duì)于SREBP1調(diào)控靶基因的促轉(zhuǎn)錄功能，通過(guò)采用CHIP-chip和CHIP-seq的研究，在人和小鼠全基因組上已發(fā)現(xiàn)上百個(gè)可能受SREBP1調(diào)控的靶基因，其中主要包括硬脂酰輔酶 A去飽和酶（stearoyl-coenzyme A desaturase,SCD）等脂肪合成酶基因[11-13]。SCD是催化飽和脂肪酸脫氫形成不飽和脂肪酸的關(guān)鍵酶，在奶牛中主要存在SCD1和SCD5兩種亞型[14]，并且SCD1在奶牛乳腺基因表達(dá)譜中所占的比例遠(yuǎn)遠(yuǎn)超過(guò)其他脂肪合成酶基因[1]。已有多個(gè)研究表明SCD1和SREBP1基因的核酸多態(tài)性均與乳脂肪性狀相關(guān)[15-16]?！颈狙芯壳腥朦c(diǎn)】目前在細(xì)胞上研究SREBP1與靶基因的轉(zhuǎn)錄調(diào)控機(jī)制多是采用間接研究模式，在培養(yǎng)細(xì)胞中加入誘導(dǎo)SREBP1的刺激因素（如胰島素/葡萄糖），通過(guò)引起SREBP1及其靶基因mRNA的表達(dá)變化來(lái)進(jìn)行推測(cè)[17]，而這些基因表達(dá)變化是SREBP1直接調(diào)控還是由于代謝通路的其他因子引起的，目前還不十分清楚[18]。還有研究采用表達(dá)SREBP1成熟核蛋白來(lái)研究其轉(zhuǎn)錄調(diào)控作用[19]，但對(duì)于SREBP1全長(zhǎng)蛋白在乳腺上皮細(xì)胞的轉(zhuǎn)錄調(diào)控機(jī)制還缺乏了解，特別是關(guān)于奶牛SREBP1在乳腺上皮細(xì)胞中調(diào)控 SCD1基因轉(zhuǎn)錄的研究還未見報(bào)道?！緮M解決的關(guān)鍵問(wèn)題】因此本研究結(jié)合前期試驗(yàn)建立的含不同調(diào)控元件奶牛 SCD1基因啟動(dòng)子[20]，通過(guò)克隆表達(dá)奶牛SREBP1蛋白，在奶牛乳腺上皮細(xì)胞中研究SREBP1對(duì)于SCD1基因啟動(dòng)子的轉(zhuǎn)錄調(diào)控作用，有助于闡明SREBP1對(duì)于靶基因的轉(zhuǎn)錄調(diào)控機(jī)制。

1 材料與方法

相關(guān)試驗(yàn)在2014年1月至2015年4月在河南農(nóng)業(yè)大學(xué)農(nóng)業(yè)部動(dòng)物生化與營(yíng)養(yǎng)重點(diǎn)實(shí)驗(yàn)室完成。

1.1 材料與儀器

奶牛乳腺組織于2014年1月采自鄭州市屠宰場(chǎng)，乳腺上皮細(xì)胞為實(shí)驗(yàn)室保存，4個(gè)不同長(zhǎng)度 pGL3-SCD1/SCD2/SCD3/SCD4熒光素酶報(bào)告基因表達(dá)載體為實(shí)驗(yàn)室構(gòu)建，所用試劑AxyPrep DNA Gel Extraction Kit 試劑盒購(gòu)自Axygen 公司，ClonExpress Multis kit試劑盒購(gòu)自Vazyme公司，Lipofectamine2000購(gòu)自Invitrogen公司，Star XL擴(kuò)增酶購(gòu)自Takala公司，雙熒光素酶檢測(cè)試劑盒購(gòu)自Promega公司，DMEM培養(yǎng)基購(gòu)自Hyclone公司，無(wú)血清培養(yǎng)基Opti-mem購(gòu)自 Gibco公司，標(biāo)簽蛋白抗體 c-myc Antibody（9E10）購(gòu)自Santa Cruz公司，熒光二抗Alexa Fluor? 488 Donkey Anti-Mouse IgG (H+L)購(gòu)自Invitrogen公司，細(xì)胞核染料DAPI、抗熒光封片淬滅液購(gòu)自碧云天，細(xì)胞培養(yǎng)板購(gòu)自 Corning公司，引物合成及測(cè)序由上海生工完成。二氧化碳培養(yǎng)箱和 Fluroskan Ascent FL熒光和化學(xué)發(fā)光檢測(cè)儀購(gòu)自 Thermo公司，激光共聚焦顯微鏡LSM 5 PASCAL購(gòu)自Carl Zeiss公司。

1.2 方法

1.2.1 奶牛SREBP1基因的克隆 采用TRIZOL提取乳腺組織的RNA，反轉(zhuǎn)錄成cDNA，根據(jù)NCBI公布的奶牛SREBP1基因序列（NM_001113302.1），設(shè)計(jì)引物時(shí)在 SREBP1序列 ATG后加入 c-myc序列（GAACAAAAACTCATCTCAGAAGAGGATCTG）作為標(biāo)簽蛋白，采用分段克隆 PCR方法對(duì)奶牛SREBP1基因進(jìn)行克隆，具體引物序列見表 1。將SREBP1總共分為四段分別進(jìn)行擴(kuò)增，同時(shí)PCR擴(kuò)增pcDNA3.1+序列作為載體序列，分別對(duì)目的基因1、2、3、4各片段純化、電泳后定量。

1.2.2 pcDNA3.1-SREBP1載體的構(gòu)建及鑒定 采用重組酶 Exnase Multis對(duì)各片段與載體進(jìn)行重組環(huán)化，重組反應(yīng)體系：5×CE Multis Buffer 4 μL，vector 108 ng，4個(gè)回收片段，Exnase Multis 2 μL，同時(shí)做無(wú)酶的空白對(duì)照，在37℃水浴30 min，置于冰水浴中反應(yīng)5 min。取反應(yīng)產(chǎn)物10 μL，加入到感受態(tài)細(xì)胞中進(jìn)行轉(zhuǎn)化，獲得克隆后挑取單菌落擴(kuò)增，提取質(zhì)粒進(jìn)行分別采用HindⅢ、XhoI雙酶切和HindⅢ、XbaI雙酶切鑒定，鑒定正確的質(zhì)粒送測(cè)序公司進(jìn)行測(cè)序。

表 1 引物序列Table 1 The sequences of primer

1.2.3 SREBP1基因mRNA在乳腺上皮細(xì)胞中的超表達(dá)將培養(yǎng)好的奶牛乳腺上皮細(xì)胞經(jīng)胰酶消化后，按比例分散培養(yǎng)在12孔細(xì)胞培養(yǎng)板中，放入37℃、5% CO2培養(yǎng)箱培養(yǎng)，到細(xì)胞達(dá)到80%的融合度時(shí)，棄去培養(yǎng)基，采用轉(zhuǎn)染試劑（Lipofectamine 2000）轉(zhuǎn)染pcDNA3.1-SREBP1質(zhì)粒6 h后，更換新鮮培養(yǎng)基，同時(shí)以pcDNA3.1空載體作為對(duì)照組，轉(zhuǎn)染48 h后提取細(xì)胞RNA，反轉(zhuǎn)錄成cDNA，采用熒光定量PCR引物（F-CCAGCTGACAGCTCCATTGA，R-TGCGCGC CACAAGGA）檢測(cè)SREBP1基因mRNA的表達(dá)，同時(shí)檢測(cè)奶牛EIF3K基因（F-CCAGGCCCACCAAGA AGAA，R- TTATACCTTCCAGGAGGTCCATGT）作為內(nèi)參基因[21]，計(jì)算SREBP1基因的mRNA表達(dá)差異倍數(shù)。

1.2.4 SREBP1蛋白在乳腺上皮細(xì)胞的定位 在24孔板中加入細(xì)胞爬片，接種乳腺上皮細(xì)胞，細(xì)胞培養(yǎng)過(guò)夜后，轉(zhuǎn)染pcDNA3.1-SREBP1質(zhì)粒，培養(yǎng)24 h后用預(yù)冷的PBS洗3遍，4%多聚甲醛固定10 min，PBS洗3遍，透化劑（0.2%Triton100）作用15 min，封閉液（90%PBS+10%胎牛血清）封閉1 h；棄封閉液，用一抗c-MYC Antibody（9E10）（1∶500）4 ℃過(guò)夜孵育；PBS洗3遍，加入熒光標(biāo)記二抗Alexa Fluor? 488 Donkey Anti-Mouse IgG（H+L）（1∶1 000）室溫避光孵育1 h；然后采用DAPI染色液（500μg·mL-1）對(duì)于細(xì)胞核染色8 min，PBS洗3遍，滴加抗熒光封片淬滅液，封片，晾干放 4℃，在激光共聚焦顯微鏡下觀察SREBP1蛋白亞細(xì)胞定位。

1.2.5 SREBP1蛋白對(duì)含不同調(diào)控元件SCD1啟動(dòng)子的轉(zhuǎn)錄調(diào)控作用 培養(yǎng)乳腺上皮細(xì)胞，以 pGL3-Basic熒光素酶空載體作為對(duì)照，轉(zhuǎn)染實(shí)驗(yàn)室前期構(gòu)建的4個(gè)含有不同調(diào)控元件的奶牛 SCD1基因啟動(dòng)子載體[20]，分別是pGL3-SCD1（212bp，序列中有TATA-box元件）、pGL3-SCD2（380bp，有CAAT-box元件）、pGL3-SCD3（416 bp，有SRE元件）和pGL3-SCD4（760 bp，有SRE、Sp1元件）（圖1），同時(shí)分別轉(zhuǎn)染1.0 μg pcDNA3.1-SREBP1質(zhì)粒作為處理因素，作用24 h后收集細(xì)胞加入裂解液，采用熒光和化學(xué)發(fā)光檢測(cè)儀進(jìn)行熒光素酶活性檢測(cè)，采用螢火蟲熒光素酶活性/海腎熒光素酶活性計(jì)算啟動(dòng)子活性，結(jié)合調(diào)控元件分析SREBP1處理對(duì)于啟動(dòng)子活性的影響。

1.2.6 SREBP1調(diào)控SCD1基因啟動(dòng)子轉(zhuǎn)錄的劑量關(guān)系 培養(yǎng)乳腺上皮細(xì)胞，接種12孔細(xì)胞培養(yǎng)板后，轉(zhuǎn)染1.0 μg pGL3-SCD2、pGL3-SCD3啟動(dòng)子載體，同時(shí)分別轉(zhuǎn)染0.25、0.5、1.0μg的pcDNA3.1-SREBP1質(zhì)粒作為處理因素，作用24 h后收集細(xì)胞，加入裂解液，進(jìn)行熒光素酶活性檢測(cè)并計(jì)算啟動(dòng)子相對(duì)活性，分析不同劑量SREBP1的處理對(duì)于啟動(dòng)子活性的影響。

圖1 含不同調(diào)控元件的奶牛SCD1啟動(dòng)子結(jié)構(gòu)Fig. 1 The structure of bovine SCD1 promoter with different regulatory elements

1.2.7 數(shù)據(jù)統(tǒng)計(jì) 試驗(yàn)中每個(gè)處理中重復(fù)4次，試驗(yàn)數(shù)據(jù)采用SPSS10.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析，**P＜0.01，***P＜0.001。

2 結(jié)果

2.1 SREBP1基因的克隆

對(duì) SREBP1基因采用分段克隆的方法，分別獲得大小約5 400 bp 的pcDNA3.1載體片段和4個(gè)不同長(zhǎng)度的目的基因片段（圖2），將各個(gè)片段切膠回收后進(jìn)行基因重組獲得連接產(chǎn)物，將連接產(chǎn)物轉(zhuǎn)化感受態(tài)細(xì)胞進(jìn)行陽(yáng)性克隆篩選，提取陽(yáng)性細(xì)菌質(zhì)粒后進(jìn)行酶切鑒定。分別采用HindIII、XhoI雙酶切，獲得3個(gè)片段，片段長(zhǎng)度分別為5 356、3 057和390 bp，采用HindIII、Xbal雙酶切，獲得2個(gè)片段，分別為5 350和3 453 bp（圖3），最后經(jīng)測(cè)序驗(yàn)證后發(fā)現(xiàn)，克隆的序列與 NCBI公布的標(biāo)準(zhǔn)序列相比，除在1 509位有一個(gè)無(wú)義突變外，其他序列均與標(biāo)準(zhǔn)序列相一致，加上標(biāo)簽蛋白myc序列，整個(gè)表達(dá)序列長(zhǎng)度達(dá)到3 510 bp。

2.2 SREBP1在乳腺上皮細(xì)胞中的超表達(dá)

將pcDNA3.1-SREBP1質(zhì)粒轉(zhuǎn)染乳腺上皮細(xì)胞后48h收集細(xì)胞，采用熒光定量PCR檢測(cè)SREBP1基因的表達(dá)豐度，結(jié)果發(fā)現(xiàn)，與轉(zhuǎn)染空載體的對(duì)照相比，SREBP1基因的mRNA表達(dá)顯著升高（圖4），比對(duì)照組增加了 130.4倍（P＜0.001），表明載體轉(zhuǎn)染后在奶牛乳腺上皮細(xì)胞中能夠高效表達(dá)SREBP1基因的mRNA。

圖2 奶牛SREBP1基因的克隆Fig. 2 The clone of bovine SREBP1

圖3 pcDNA3.1-SREBP1質(zhì)粒酶切鑒定圖Fig. 3 The restriction enzyme digestion of pcDNA3.1-SREBP1

2.3 SREBP1蛋白在乳腺上皮細(xì)胞中的定位

將 pcDNA3.1-SREBP1載體轉(zhuǎn)染乳腺上皮細(xì)胞后，采用激光共聚焦顯微鏡觀察發(fā)現(xiàn)（圖 5），進(jìn)行免疫熒光標(biāo)記的SREBP1發(fā)出綠色熒光，細(xì)胞核經(jīng) DAPI染色后發(fā)出藍(lán)色熒光，共定位發(fā)現(xiàn)綠色和藍(lán)色融合形成一種青色熒光定位在細(xì)胞核位置，表明SREBP1作為轉(zhuǎn)錄因子能夠轉(zhuǎn)運(yùn)到乳腺上皮細(xì)胞核中進(jìn)行表達(dá)。

2.4 SREBP1蛋白對(duì)于不同SCD1啟動(dòng)子的調(diào)控作用

將含不同調(diào)控元件的 SCD1基因啟動(dòng)子 pGL3-SCD1、SCD2、SCD3和SCD4轉(zhuǎn)染細(xì)胞后，同時(shí)轉(zhuǎn)染SREBP1質(zhì)粒進(jìn)行處理，檢測(cè)啟動(dòng)子活性發(fā)現(xiàn)（圖6），對(duì)照組basic啟動(dòng)子活性為2.32，在SREBP1刺激后啟動(dòng)子活性降低到1.29（P＜0.01），與此類似，SCD1和SCD2啟動(dòng)子進(jìn)行SREBP1處理后，熒光值均顯著降低（P＜0.01），而對(duì) SCD3、SCD4啟動(dòng)子進(jìn)行SREBP1處理后，啟動(dòng)子活性均有顯著增加。其中SCD3啟動(dòng)子活性由59.92增加到124.37，增加了1.0倍（P＜0.001），SCD4進(jìn)行SREBP1處理后，啟動(dòng)子活性由 83.55增加到 145.48，增加了 0.7倍（P＜ 0.001），表明SREBP1蛋白表達(dá)后，顯著增加了SCD3、SCD4啟動(dòng)子的活性，其中對(duì)于SCD3啟動(dòng)子的活性促進(jìn)最為明顯。

圖4 SREBP1基因mRNA在乳腺上皮細(xì)胞中的表達(dá)豐度Fig. 4 The mRNA expression of SREBP1 in mammary epithelial cell (n=4)

圖5 SREBP1蛋白在乳腺上皮細(xì)胞的表達(dá)定位Fig. 5 The localization of SREBP1 in mammary epithelial cell

圖6 SREBP1對(duì)不同SCD1啟動(dòng)子活性的調(diào)控Fig. 6 The regulation of different SCD1 promoter activities by SREBP1

2.5 SREBP1調(diào)控SCD基因啟動(dòng)子轉(zhuǎn)錄的量效關(guān)系

圖7 不同含量SREBP1對(duì)pGL3-SCD2、3啟動(dòng)子活性的調(diào)控Fig. 7 The regulation of pGL3-SCD2 and SCD3 promoter activities by different doses of SREBP1

在轉(zhuǎn)染0.25、0.5、1.0 μg的 SREBP1質(zhì)粒后，pGL3-SCD2啟動(dòng)子的活性呈現(xiàn)持續(xù)下降的趨勢(shì)(圖7)，從19.90降低到10.31，而pGL3- SCD3啟動(dòng)子的活性隨著質(zhì)粒含量的增加則呈現(xiàn)上升趨勢(shì)，其活性值從59.81分別增加到70.76、101.48和108.43（P＜0.001），表明SREBP1能夠劑量依賴性的增強(qiáng)pGL3- SCD3啟動(dòng)子的活性。

3 討論

固醇調(diào)節(jié)元件結(jié)合蛋白（SREBP）屬于“堿性螺旋-環(huán)-螺旋亮氨酸拉鏈”轉(zhuǎn)錄因子家族，其中SREBP1蛋白結(jié)構(gòu)域一般分為3部分，包括480個(gè)氨基酸的N-端，80個(gè)氨基酸的跨膜結(jié)構(gòu)域和590個(gè)氨基酸的C-端[22-23]。本研究中荷斯坦奶牛SREBP1基因的編碼區(qū)全長(zhǎng)3 440 bp，加上標(biāo)簽蛋白序列后整個(gè)基因長(zhǎng)度在3 510 bp左右。由于SREBP1基因序列中含有復(fù)雜的二級(jí)結(jié)構(gòu)，為提高克隆的準(zhǔn)確性和效率，本研究采用分段克隆、基因重組的方法，得到了完整的SREBP1基因編碼區(qū)序列，表明此分段克隆重組的方法對(duì)于長(zhǎng)片段的基因克隆具有較好的效果。經(jīng)過(guò)細(xì)胞轉(zhuǎn)染和熒光定量PCR檢測(cè)發(fā)現(xiàn)，SREBP1基因的mRNA有顯著表達(dá)（圖4），表明載體構(gòu)建成功。

SREBP1作為在內(nèi)質(zhì)網(wǎng)合成的蛋白質(zhì)，首先合成無(wú)活性的前體（大約1 150個(gè)AA），然后通過(guò)與SCAP的相互作用后進(jìn)入到高爾基體進(jìn)行水解活化，暴露出NH2-末端活性結(jié)構(gòu)域形成入核蛋白（大約500個(gè)AA），進(jìn)入到細(xì)胞核內(nèi)與靶基因啟動(dòng)子結(jié)合調(diào)控轉(zhuǎn)錄[24]。因此本研究采用激光共聚焦對(duì)表達(dá)的SREBP1蛋白進(jìn)行亞細(xì)胞定位后，發(fā)現(xiàn)SREBP1能夠在細(xì)胞核中表達(dá)（圖5），這說(shuō)明構(gòu)建的 SREBP1質(zhì)粒在細(xì)胞內(nèi)表達(dá)全長(zhǎng)蛋白后能夠水解活化形成入核蛋白，轉(zhuǎn)運(yùn)到細(xì)胞核內(nèi)發(fā)揮功能。

對(duì)于在乳腺上皮細(xì)胞中 SREBP1與靶基因的研究發(fā)現(xiàn)，SREBP1通過(guò)激活PⅢ啟動(dòng)子來(lái)調(diào)節(jié)乙酰輔酶A羧化酶基因的轉(zhuǎn)錄[25]。MA等在乳腺上皮細(xì)胞中通過(guò)構(gòu)建SREBP1的siRNA抑制載體，能夠降低SCD、ACC和FAS等多種脂肪酸合成酶基因mRNA的表達(dá)[26]。本研究采用不同調(diào)控元件的 SCD1啟動(dòng)子，通過(guò)在乳腺上皮細(xì)胞表達(dá)SREBP1后發(fā)現(xiàn)pGL3-SCD1-2啟動(dòng)子的活性顯著降低，而 pGL3-SCD3-4啟動(dòng)子的活性顯著增加（圖6）。研究表明SREBPs 自身是非常低效的轉(zhuǎn)錄因子, 只有接近結(jié)合位點(diǎn)時(shí)才能刺激靶基因的表達(dá)[27]，而 pGL3-SCD3、4比pGL3-SCD1、2在結(jié)構(gòu)上主要多出了一個(gè)SRE調(diào)控元件（圖1），表明SRE調(diào)控元件可能對(duì)于SREBP1調(diào)控 SCD1基因轉(zhuǎn)錄具有重要作用。ZULKIFLI在HEK細(xì)胞中采用表達(dá)人 SREBP1-c成熟核蛋白來(lái)調(diào)控羊SCD基因啟動(dòng)子的轉(zhuǎn)錄，發(fā)現(xiàn)去除羊SCD基因啟動(dòng)子的SRE元件顯著降低了啟動(dòng)子活性，其結(jié)果與本研究一致[19]。為了進(jìn)一步驗(yàn)證SRE調(diào)控元件的作用，試驗(yàn)又采用不同濃度的 SREBP1刺激 pGL3-SCD2、pGL3-SCD3啟動(dòng)子，結(jié)果發(fā)現(xiàn) pGL3-SCD3啟動(dòng)子的活性增加與 SREBP1的濃度呈現(xiàn)劑量依賴性，結(jié)合pGL3-SCD3與pGL3-SCD2啟動(dòng)子結(jié)構(gòu)上相比只有36bp的差異（其中含有SRE調(diào)控元件），說(shuō)明SREBP1可以通過(guò)與SRE元件結(jié)合促進(jìn)SCD1基因轉(zhuǎn)錄。

啟動(dòng)子上與轉(zhuǎn)錄因子結(jié)合的位點(diǎn)具有特定模式,稱為模體，一般長(zhǎng)度在5—20 bp范圍內(nèi)。轉(zhuǎn)錄因子在細(xì)胞內(nèi)可以同時(shí)調(diào)控多個(gè)基因，而在不同基因上的結(jié)合位點(diǎn)并不完全相同。研究認(rèn)為SREBP1結(jié)合的經(jīng)典模體為（5′-TCACNCCAC-3′）/E-和（5′-CANNTG-3′）兩種[13,18]。本研究中 SRE調(diào)控元件含有的模體為5′-AGCAGATTGCG-3′（圖1），與經(jīng)典的SREBP1模體序列并不相符。TABOR 等在HepG2細(xì)胞上研究小鼠SREBP1a與鼠SCD基因啟動(dòng)子的關(guān)系，發(fā)現(xiàn)了與本研究一致的SRE新模體（5′-AGCAGATTG CG-3′）對(duì)于啟動(dòng)子活性至關(guān)重要，而經(jīng)典模體反而對(duì)啟動(dòng)子活性沒(méi)有顯著影響[28]，ZULKIFLI比對(duì)人、豬、鼠、羊的SCD1基因啟動(dòng)子序列后發(fā)現(xiàn)此模體在不同物種間是高度保守的[19]。這些結(jié)果說(shuō)明SREBP1可能通過(guò)與SCD啟動(dòng)子上（5′-AGCAGAT TGCG-3′）模體的結(jié)合，發(fā)揮了轉(zhuǎn)錄因子的促轉(zhuǎn)錄作用。SEO等在小鼠組織中曾經(jīng)發(fā)現(xiàn)了 1 個(gè)新的SREBP1模體，并認(rèn)為是經(jīng)典模體的一個(gè)功能變體[12]，因此本研究中發(fā)現(xiàn)的 SREBP1模體序列可能是SCD1基因所特有的一個(gè)功能變體，這還需要進(jìn)一步的研究證實(shí)。

4 結(jié)論

本研究通過(guò)克隆構(gòu)建奶牛固醇調(diào)節(jié)元件結(jié)合蛋白1真核表達(dá)載體，發(fā)現(xiàn)固醇調(diào)節(jié)元件結(jié)合蛋白1能夠定位在乳腺上皮細(xì)胞核中，并且固醇調(diào)節(jié)元件結(jié)合蛋白1可以與SRE調(diào)控元件結(jié)合促進(jìn)SCD1基因啟動(dòng)子的轉(zhuǎn)錄。

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（責(zé)任編輯 林鑒非）

Expression and Localization of Bovine SREBP1 Protein and Regulation of the Transcription of SCD1 Promoter in Bovine Mammary Epithelial Cell

HAN Li-qiang, WANG Yue-ying, WANG Lin-feng, ZHU He-shui, ZHONG Kai, CHU Bei-bei, YANG Guo-yu
(College of Animal Science and Veterinary Medicine, Henan Agricultural University,Key Laboratory of Animal Biochemistry and Nutrition of Ministry of Agriculture, Zhengzhou 450002)

bovine; SREBP1; mammary epithelial cell; subcellular localization; gene regulation

ntia Agricultura Sinica

10.3864/j.issn.0578-1752.2016.24.011

2015-07-29；接受日期：2016-10-17

國(guó)家“973”項(xiàng)目（2011CB100802）、國(guó)家自然科學(xué)基金（31072009）、國(guó)家轉(zhuǎn)基因重大專項(xiàng)（2014ZX0801015B）、河南省高等學(xué)校重點(diǎn)科研項(xiàng)目（15A230020）、河南省基礎(chǔ)與前沿項(xiàng)目（162300410258）

聯(lián)系方式：韓立強(qiáng)，E-mail：qlhan2001@126.com。通信作者楊國(guó)宇，E-mail：haubiochem@163.com