彭躍躍丁 燏,王金慧簡(jiǎn)紀(jì)常,魯義善,劉 瑩

(1. 廣東海洋大學(xué)水產(chǎn)學(xué)院, 湛江 524025; 2. 廣東省水產(chǎn)經(jīng)濟(jì)動(dòng)物病原生物學(xué)及流行病學(xué)重點(diǎn)實(shí)驗(yàn)室, 湛江 524025; 3. 廣東省教育廳水產(chǎn)經(jīng)濟(jì)動(dòng)物病害控制重點(diǎn)實(shí)驗(yàn)室, 湛江 524025)

南極衣藻谷胱甘肽-S-轉(zhuǎn)移酶基因的表達(dá)及其對(duì)大腸桿菌低溫耐受性的增強(qiáng)作用

彭躍躍1,2丁 燏1,2,3王金慧1,2簡(jiǎn)紀(jì)常1,2,3魯義善1,2,3劉 瑩1,2

(1. 廣東海洋大學(xué)水產(chǎn)學(xué)院, 湛江 524025; 2. 廣東省水產(chǎn)經(jīng)濟(jì)動(dòng)物病原生物學(xué)及流行病學(xué)重點(diǎn)實(shí)驗(yàn)室, 湛江 524025; 3. 廣東省教育廳水產(chǎn)經(jīng)濟(jì)動(dòng)物病害控制重點(diǎn)實(shí)驗(yàn)室, 湛江 524025)

谷胱甘肽-S-轉(zhuǎn)移酶(Glutathione-S-transferase, GST, EC2.5.1.18)是生物體內(nèi)一種重要的抗氧化酶, 為闡明GST在南極衣藻(Chlamydomonas sp. ICE-L)中的具體地位, 采用實(shí)時(shí)熒光定量PCR對(duì)不同溫度下南極衣藻的GST基因的表達(dá)進(jìn)行了分析; 并構(gòu)建了原核表達(dá)載體pET28a(+)-GST, 轉(zhuǎn)化至大腸桿菌BL21(DE3)中進(jìn)行誘導(dǎo)表達(dá), 通過(guò)平板培養(yǎng)法探討了重組菌E. coli BL21(pET28a(+)-GST)對(duì)低溫脅迫的耐受性。結(jié)果顯示, GST在0℃時(shí)表達(dá)量最高, 最高可達(dá)對(duì)照組的兩倍多; pET28a(+)-GST重組表達(dá)載體在E. coli BL21中實(shí)現(xiàn)了高效表達(dá), 且主要以包涵體形式存在, 經(jīng)HisTrap HP柱分離純化獲得高純度的GST融合蛋白, 并通過(guò)SDS-PAGE及Western blot分析得以驗(yàn)證; 對(duì)低溫脅迫實(shí)驗(yàn)發(fā)現(xiàn)南極衣藻GST蛋白的表達(dá)可以提高重組菌E. coli BL21對(duì)低溫的耐受性, 說(shuō)明GST基因?qū)δ蠘O衣藻適應(yīng)南極低溫環(huán)境具有重要作用。

南極衣藻; 谷胱甘肽-S-轉(zhuǎn)移酶; 表達(dá); 低溫

谷胱甘肽-S-轉(zhuǎn)移酶(GST: EC2.5.1.18)是一種多功能酶, 廣泛存在于各種動(dòng)物、植物及微生物中, 分子量主要介于23—29 kD之間。根據(jù)其基因組織結(jié)構(gòu)和序列的相似性, GSTs分為6類(lèi): phi、tau、lambda、theta和zeta及脫氫抗壞血酸還原酶(DHARs)[1], 前3類(lèi)是植物所特有, 主要在植物的初級(jí)代謝和次級(jí)代謝、脅迫耐受和細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中起作用[2], 其中在各種脅迫中主要通過(guò)清除因脅迫產(chǎn)生的過(guò)氧化物(過(guò)氧化氫、過(guò)氧酸鹽)等物質(zhì)起作用[3]。已有的研究表明, GST的過(guò)量表達(dá)可以增強(qiáng)蘆葦(Phragmites australis)對(duì)Cd2+的耐受性, 并具有阻止細(xì)胞凋亡的作用[4]; 在擬南芥(Arabidopsis)中GST在多種脅迫(如傷害[5]、乙烯利[6]、生長(zhǎng)素[7]、過(guò)氧化氫(H2O2)和水楊酸[8])應(yīng)答中均起著重要的作用; 梨形環(huán)棱螺肝臟和鰓的GST酶活性受一定濃度Cu2+的誘導(dǎo)作用[9]。 GST對(duì)植物適應(yīng)低溫環(huán)境也有一定的作用, 橡膠樹(shù)(Hevea brasiliensis)GST基因的表達(dá)明顯受低溫的調(diào)控[10]; 冷脅迫能夠在轉(zhuǎn)錄和翻譯水平上引起甘薯(Ipomoea batats. L)GST的響應(yīng), 協(xié)助保護(hù)組織免受傷害[11]; 生長(zhǎng)于寒冷環(huán)境中野生馬鈴薯(Solanum commersonii)GST基因的表達(dá)量比低溫敏感馬鈴薯高[12]; 云杉卷葉蛾(Choristoneura fumiferana)GST基因在轉(zhuǎn)基因擬南芥中的表達(dá)可以增強(qiáng)擬南芥對(duì)低溫的耐受性[13]; GST基因的過(guò)量表達(dá)可以促進(jìn)轉(zhuǎn)基因水稻在低溫中的發(fā)芽和生長(zhǎng)[14]。南極衣藻(Chlamydomonas sp. ICE-L)是生活在南極海冰表面、內(nèi)部和底部一類(lèi)微型單胞藻類(lèi), 嚴(yán)寒是南極環(huán)境一個(gè)最基本的特征。前期研究發(fā)現(xiàn)南極衣藻谷胱甘肽相關(guān)酶對(duì)其適應(yīng)南極嚴(yán)寒環(huán)境具有一定的作用[15], 在蛋白水平上南極衣藻GST的活性與溫度密切相關(guān)[16]。本研究旨在進(jìn)一步從分子水平上分析南極衣藻GST基因表達(dá)與溫度的關(guān)系, 同時(shí)構(gòu)建了pET- 28a(+)-GST重組表達(dá)載體, 從原核水平上檢測(cè)南極衣藻GST基因的表達(dá)能否提高宿主菌E. coli BL21對(duì)低溫的耐受性, 為后續(xù)關(guān)于GST在抗低溫及其他抗逆研究中的應(yīng)用奠定理論基礎(chǔ)。

1 材料與方法

1.1 材料與試劑

RNA提取試劑TRIZOL?Reagent購(gòu)自Invitrogen公司, dNTPs、ExTaq酶、rTaq酶及Reverse Transcriptase M-MLV(Rnase H-)均購(gòu)自TaKaRa公司, TransStartTMGreen qPCR SuperMix購(gòu)自北京全式金公司, 異丙基-β-D-硫代吡喃半乳糖苷(IPTG)購(gòu)自上海生工生物技術(shù)有限公司, 鼠抗His·tag單抗、辣根過(guò)氧化物酶標(biāo)記的山羊抗鼠IgG及DAB顯色試劑盒均購(gòu)自武漢博士德生物工程有限公司, NC膜購(gòu)于PALL公司。

本實(shí)驗(yàn)對(duì)象南極衣藻(Chlamydomonas sp. ICE-L)由國(guó)家海洋局第一海洋研究所海洋生物活性物質(zhì)重點(diǎn)實(shí)驗(yàn)室提供。E. coli BL21(DH5α)、pET-28a(+)質(zhì)粒由廣東海洋大水產(chǎn)學(xué)院廣東省水產(chǎn)經(jīng)濟(jì)動(dòng)物病原生物學(xué)及流行病學(xué)重點(diǎn)實(shí)驗(yàn)室保存。

1.2 GST在不同溫度下的定量分析

采用Provasoli培養(yǎng)基培養(yǎng)南極衣藻, 溫度為4—8℃, 光強(qiáng)為1300—1900 lx, 光照周期為12h∶12h, 不充氣, 每天搖3—4次。將藻培養(yǎng)液分別置于0℃、8℃、14℃培養(yǎng), 各溫度組均設(shè)3個(gè)平行, 并在0、6、12、24、36、48、72h分別收集藻體。按TRIZOL?Reagent試劑的操作程序稍加改動(dòng)提取總RNA, 經(jīng)DNase I消化后用Reverse Transcriptase M-MLV (Rnase H?)和oligodT18按照M-MLV說(shuō)明書(shū)合成cDNA一鏈。以β-action為內(nèi)參[17], 進(jìn)行熒光定量PCR擴(kuò)增。取2 μL上述稀釋20倍后的cDNA一鏈作為模板, 反應(yīng)體系25 μL包括: 2×TransStart Green qPCR SuperMix 12.5 μL和10 mmol/L的上下引物各1 μL以及8.5 μL ddH2O, PCR反應(yīng)程序?yàn)? 94℃預(yù)變性5min, 94℃ 20s, 58℃ 20s, 72℃ 20s,進(jìn)行40個(gè)循環(huán), 最后72℃延伸30s。應(yīng)用iQTM5 Optical System Software, 采用2?ΔΔCt法進(jìn)行目標(biāo)基因的熒光定量分析, 應(yīng)用SPSS 17.0軟件對(duì)獲得的實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析, **表示與同期對(duì)照組相比差異極顯著(P<0.01), *表示與同期對(duì)照組相比差異顯著(0.01< P<0.05)。

1.3 pE T-28a(+)-GST原核表達(dá)載體的構(gòu)建、表達(dá)以及條件的優(yōu)化

根據(jù)本實(shí)驗(yàn)室在GenBank所提交的南極衣藻GST基因序列(登錄號(hào): HQ444265)設(shè)計(jì)特異引物對(duì)其開(kāi)放閱讀框(ORF)進(jìn)行擴(kuò)增。F1: 5′-CGCGGATCC ATGCGAGCTGCTCTGCGT-3′(BamH I), R1: 5′-ACGC GTCGAC CTACGTAGCCCTTGATGCCA-3′(Sal I), 在上、下游引物分別引入BamH I和Sal I酶切位點(diǎn)。以cDNA一鏈為模板進(jìn)行PCR(94℃ 5min, 94℃ 30s, 60℃ 30s, 72℃ 1min, 72℃ 5min)擴(kuò)增, PCR產(chǎn)物和空質(zhì)粒pET-28a(+)分別用BamH I、Sal I酶切, 并回收目的片段和線(xiàn)性化載體, T4DNA連接酶16℃連接過(guò)夜, 構(gòu)建的表達(dá)載體pET-28a(+)-GST轉(zhuǎn)化至BL21(DE3)中。陽(yáng)性克隆經(jīng)酶切鑒定后送上海生工測(cè)序驗(yàn)證。

將含有pET-28a(+)-GST的BL21接種于含50 μg/mL卡那霉素(Kan+)的LB培養(yǎng)液中, 37℃振蕩培養(yǎng)過(guò)夜, 將此培養(yǎng)物按1∶50的體積比添加到LB(Kan+)中, 分別在18℃、28℃、37℃培養(yǎng)至A6000.4—0.6, 加入IPTG至終濃度為1 mmol/L, 誘導(dǎo)4h后, 取菌液10 mL, 10000 r/min離心1min, 加入500 μL 1×PBS(KH2PO40.2 g, Na2HPO4·12H2O 2.9 g, NaCl 8.0 g, KCl 0.2 g, pH 7.4)重懸沉淀后冰浴超聲波破碎,離心收集上清和沉淀, 沉淀用1×PBS重懸浮, 分別吸取20 μL上清和重懸浮沉淀加入2×上樣緩沖液,煮沸5min, 進(jìn)行12% SDS-PAGE電泳分析。對(duì)IPTG濃度的優(yōu)化, 37℃下培養(yǎng)重組菌至A6000.4—0.6時(shí)加入IPTG至終濃度分別為0、0.2、0.4、0.6、0.8、1.0、1.2 mmoL/L進(jìn)行誘導(dǎo)4h; 對(duì)于時(shí)間的優(yōu)化, 添加同濃度的IPTG誘導(dǎo)0、1、2、3、4、5、6h后分別取樣分別進(jìn)行12%SDS-PAGE電泳分析。

1.4 pE T-28a(+)-GST表達(dá)蛋白的純化以及western blot鑒定

按最佳誘導(dǎo)條件大量誘導(dǎo)(100 mL)重組表達(dá)蛋白, 離心收集菌體, 1×PBS重懸菌體, 超聲破碎, 工作時(shí)間8s, 間隔9s, 30min。離心收集沉淀, 分別用Washing buffer (Triton-100 0.5%, Tris-HCl 50 mmol/L pH 8.0, NaCl 300 mmol/L, EDTA 10 mmol/L, DTT 10 mmol/L)洗滌兩次、Resuspension buffer (Tris-HCl 50 mmol/L pH 8.0, NaCl 100 mmol/L, EDTA 10 mmol/L, DTT 10mmol/L)洗滌一次, 離心收集沉淀用8 mol/L尿素過(guò)夜溶解沉淀。離心收集上清用HisTrap HP 1 mL進(jìn)行純化。純化蛋白經(jīng)SDS-PAGE電泳后, 然后進(jìn)行蛋白質(zhì)電轉(zhuǎn)移, 將凝膠上的蛋白轉(zhuǎn)印至硝酸纖維素膜上(轉(zhuǎn)移條件100V, 150min), 轉(zhuǎn)移完畢膜用5%脫脂奶粉Tris緩沖鹽-吐溫溶液(TTBS)室溫封閉過(guò)夜; 次日用TTBS緩沖液洗滌3次后用鼠抗His·tag單抗37℃孵育1h; 再用TTBS洗膜3次, 再用辣根過(guò)氧化物酶標(biāo)記山羊抗小鼠IgG37℃孵育1h;再用TTBS洗膜3次, 用DAB顯色至目的條帶清晰后立即以ddH2O終止反應(yīng)。

1.5 重組菌BL21(pET-28a(+)-GST)的耐低溫分析

將重組菌E. coli BL21(pET-28a(+)-GST)接種于LB(Kan+)液體培養(yǎng)基中培養(yǎng), 當(dāng)A600為0.4—0.6時(shí),加入IPTG至終濃度為0.2 mmol/L, 150 r/min誘導(dǎo)培養(yǎng)6h。取上述培養(yǎng)物于無(wú)菌試管中, 稀釋105后各取100 μL涂于LB(Kan+)平板。4℃分別處理0、1、2、3、4、5、6d后, 置于37℃繼續(xù)培養(yǎng)過(guò)夜并統(tǒng)計(jì)菌落數(shù), 同時(shí)以BL21 [pET-28a(+)]菌株作為對(duì)照。每個(gè)處理均設(shè)置3個(gè)重復(fù), 結(jié)果用統(tǒng)計(jì)軟件SPSS(17.0)進(jìn)行分析, **表示與同期對(duì)照組相比差異極顯著(P<0.01), *表示與同期對(duì)照組相比差異顯著(0.01

2 結(jié)果

2.1 南極衣藻GST的定量表達(dá)

實(shí)時(shí)熒光定量PCR結(jié)果顯示, 在3個(gè)溫度GST均有表達(dá)(圖1), 且在對(duì)照溫度8℃時(shí)各個(gè)取樣時(shí)間GST的表達(dá)量相當(dāng); 0℃時(shí), 第6h表達(dá)量低于初始水平, 第12h迅速恢復(fù)并繼續(xù)上升, 在處理36h時(shí)表達(dá)量達(dá)到最大, 為正常水平的兩倍多。其中在12h、24h時(shí)表達(dá)量顯著高于對(duì)照(P<0.05), 36h時(shí)表達(dá)量極顯著高于對(duì)照(P<0.01); 14℃時(shí), GST的表達(dá)量均低于對(duì)照組, 且隨著時(shí)間的推移有一個(gè)緩慢下降的趨勢(shì), 72h時(shí)的表達(dá)量約只有對(duì)照組的四分之一。

圖1 南極衣藻GST在不同溫度下的表達(dá)Fig. 1 The Real-time PCR analysis of the expression level of Chlamydomonas sp. ICE-L GST at different temperature

圖2 重組表達(dá)載體pET-28a(+)-GST的酶切鑒定Fig. 2 Restriction analysis of recombinant expression plasmid pET-28a(+)-GST

2.2 pE T-28a(+)-GST原核表達(dá)載體的構(gòu)建

南極衣藻GST開(kāi)放閱讀框PCR產(chǎn)物經(jīng)測(cè)序證明GST-ORF擴(kuò)增成功。重組表達(dá)載體(pET-28a (+)-GST)經(jīng)BamH I、Sal I雙酶切, 呈現(xiàn)目的條帶和載體片段大小(圖2), 且測(cè)序結(jié)果共同證明重組表達(dá)載體構(gòu)建成功。誘導(dǎo)表達(dá)實(shí)驗(yàn)結(jié)果(圖3)顯示, pET-28a (+)-GST重組表達(dá)載體在18℃、28℃、37℃均能表達(dá), 并在37℃誘導(dǎo)時(shí)表達(dá)量最大, 表達(dá)蛋白主要以包涵體形式存在;在最佳誘導(dǎo)溫度37℃時(shí),目的蛋白的表達(dá)量受IPTG濃度影響較小, 但是無(wú)IPTG誘導(dǎo)目的蛋白不表達(dá); 在一定時(shí)間內(nèi), 經(jīng)相同IPTG濃度誘導(dǎo)目的蛋白的表達(dá)量隨時(shí)間的增加而增加, 在4h時(shí)達(dá)到最大。

圖3 重組表達(dá)載體pET-28a(+)-GST表達(dá)的優(yōu)化Fig. 3 The optimization of pET-28a(+)-GST expression

2.3 pE T-28a(+)- GST表達(dá)蛋白的純化及Western blot鑒定

表達(dá)載體經(jīng)0.2 mmol/L IPTG、37℃誘導(dǎo)4h獲得足量的表達(dá)產(chǎn)物, 經(jīng)前期處理后采用HisTrap HP層析柱分離純化, SDS-PAGE電泳顯示得到的蛋白分子量約為26 kD(圖4B); Western blot檢測(cè),在26 kD左右也有明顯條帶(圖4A),證明表達(dá)的蛋白為南極衣藻谷胱甘肽硫轉(zhuǎn)移酶蛋白。

圖4 pET-28a(+)-GST融合蛋白的SDSPAGE分析(B)和Western blot分析(A)Fig. 4 pET-28a(+)-GST purification product analysis of fusion protein by SDSPAGE (B) and Western blot analysis (A) M. protein marker 1, 2. GST expressed protein

2.4 重組菌BL21(pET-28a(+)-GST)的低溫耐受性分析

重組菌在LB板(Kan+)上經(jīng)4℃處理不同時(shí)間后的生長(zhǎng)結(jié)果(圖5)表明, 重組菌BL21(pET-28a(+)-GST)的存活率普遍高于對(duì)照菌BL21(pET-28a(+))。且在4天內(nèi), 重組菌的存活率顯著高于對(duì)照組(P<0.05), 說(shuō)明重組菌的耐低溫能力得到增強(qiáng); 而在第5、第6天時(shí)重組菌BL21(pET-28a(+)-GST)和對(duì)照菌BL21(pET-28a(+))存活率比較接近, 對(duì)低溫的耐受性沒(méi)有顯著性的差異(P>0.05)。

圖5 GST重組表達(dá)后宿主菌E. coli的耐低溫性分析Fig. 5 Protective effect of recombinant pET-28a(+)-GST on BL21 cell viability subjected to 4℃ treatment

3 討論

南極是地球上最冷的地區(qū), 水溫常年在0℃左右, 低溫被認(rèn)為是南極生物生存的一個(gè)最大挑戰(zhàn)。在低溫等環(huán)境中, 植物通常會(huì)產(chǎn)生一系列的氧化反應(yīng)產(chǎn)生活性氧, 導(dǎo)致氧化脅迫, 對(duì)細(xì)胞造成很強(qiáng)的破壞性。GST作為生物體內(nèi)的主要的抗氧化酶之一,可以催化谷胱甘肽與過(guò)氧化物質(zhì)發(fā)生反應(yīng), 及時(shí)消除因嚴(yán)寒所產(chǎn)生的氧化脅迫, 將其轉(zhuǎn)化為無(wú)害或低毒物質(zhì), 從而協(xié)助保護(hù)生物體免受損害[18]。研究枸橘對(duì)低溫脅迫響應(yīng)基因時(shí), 發(fā)現(xiàn)4℃時(shí)的GST酶活性是25℃時(shí)的1.5倍[19]; 丁燏等[16]在蛋白水平上研究了溫度對(duì)南極衣藻中谷胱甘肽相關(guān)酶活性的影響,發(fā)現(xiàn)低溫(0℃、?10℃)時(shí)GST的活性明顯高于對(duì)照組; Sepp?nen, et al.[12]研究了生長(zhǎng)于寒冷環(huán)境中野生馬鈴薯中GST基因的特性與表達(dá)發(fā)現(xiàn), 該物種GST在寒冷環(huán)境中的表達(dá)量明顯增強(qiáng), 說(shuō)明寒冷能促進(jìn)該物種GST基因的表達(dá)。本文根據(jù)南極衣藻在實(shí)驗(yàn)室的最佳培養(yǎng)溫度為4—8℃, 應(yīng)用實(shí)時(shí)熒光定量PCR檢測(cè)了南極衣藻經(jīng)0℃與14℃處理后GST基因的表達(dá)情況(8℃為對(duì)照), 發(fā)現(xiàn)低溫時(shí)GST表達(dá)量最高, 最高時(shí)可達(dá)到對(duì)照組的2倍多、高溫組的5倍多(圖1), 在丁燏等[16]對(duì)南極衣藻經(jīng)不同溫度處理后, 提取GST蛋白測(cè)其活性研究中發(fā)現(xiàn): 低溫處理后GST酶活性較高, 最高可達(dá)到對(duì)照組的4倍; 說(shuō)明低溫時(shí)GST酶的活性升高可能與GST的表達(dá)量的增加有關(guān)。從而本研究結(jié)果從基因表達(dá)水平進(jìn)一步證實(shí)了丁燏等的研究結(jié)果, 證實(shí)低溫能誘導(dǎo)南極衣藻GST的表達(dá)以抵抗嚴(yán)寒環(huán)境所產(chǎn)生的氧化脅迫,從而幫助其有效地適應(yīng)低溫環(huán)境。本文的結(jié)果也與其他植物的研究一致[12,19], 可見(jiàn)GST在生物適應(yīng)低溫環(huán)境中具有普遍的作用。

為了進(jìn)一步驗(yàn)證南極衣藻適應(yīng)南極嚴(yán)寒環(huán)境時(shí)GST所起的作用, 我們利用大腸桿菌表達(dá)體系具有培養(yǎng)周期短、成本低、高效表達(dá)和帶有His·tag易于純化等的優(yōu)點(diǎn)[20], 構(gòu)建了pET-28a(+)-GST表達(dá)載體(圖2)。由于IPTG濃度對(duì)pET-28a(+)-GST蛋白的誘導(dǎo)表達(dá)影響不明顯, 根據(jù)IPTG濃度過(guò)高會(huì)抑制細(xì)菌生長(zhǎng)的原則, 確定重組質(zhì)粒最佳表達(dá)條件: 0.2 mmol/L IPTG、37℃誘導(dǎo)4h。表達(dá)條件的優(yōu)化為后續(xù)獲得大量GST蛋白以進(jìn)行更深入的研究打下了基礎(chǔ)。

在南極衣藻GST的表達(dá)對(duì)E. coli BL21低溫耐受性增強(qiáng)實(shí)驗(yàn)中, 由于表達(dá)產(chǎn)物主要以包涵體形式存在, 研究發(fā)現(xiàn)在原核表達(dá)產(chǎn)物中包涵體的形成主要是由于蛋白的表達(dá)量高、合成速度太快, 致使蛋白沒(méi)有足夠的時(shí)間進(jìn)行正確的折疊, 過(guò)多的蛋白間的非特異性結(jié)合, 蛋白質(zhì)無(wú)法達(dá)到足夠的溶解度所形成, 而低表達(dá)時(shí)很少形成包涵體[21]。為了讓目的蛋白GST在E. coli BL21內(nèi)緩慢表達(dá)以減少包涵體的形成, 我們采取了降低轉(zhuǎn)速、延長(zhǎng)誘導(dǎo)時(shí)間等措施。耐低溫實(shí)驗(yàn)結(jié)果顯示, 在低溫條件下, 含有重組表達(dá)質(zhì)粒的E. coli BL21的存活率在一定時(shí)間內(nèi)比對(duì)照菌高, 說(shuō)明南極衣藻GST在大腸桿菌E. coli BL21中的表達(dá)在一定時(shí)間內(nèi)可以提高大腸桿菌的耐低溫能力。該結(jié)果與本研究熒光定量PCR結(jié)果及前人關(guān)于南極衣藻GST相關(guān)研究結(jié)果一致, 進(jìn)一步驗(yàn)證了南極衣藻GST基因的表達(dá)對(duì)其適應(yīng)南極低溫環(huán)境起到了一定的積極作用。

近年來(lái), 植物的抗逆性研究逐漸成為一個(gè)熱點(diǎn),本研究結(jié)果證明了南極衣藻GST在其抗寒方面具有重要的作用, 這為了解南極衣藻對(duì)南極寒冷環(huán)境的適應(yīng)提供了重要的理論依據(jù), 也為植物抗逆基因工程應(yīng)用中提供了重要的資料。

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INCREASED ESCHERICHIA COLI TOLERANCE TO LOW TEMPERATURE BY EXPRESSION OF GLUTATHIONE-S-TRANSFERASE GENE FROM THE CHLAMYDOMONAS SP. ICE-L

PENG Yue-Yue1,2, DING Yu1,2,3, WANG Jin-Hui1,2, JIAN Ji-Chang1,2,3, LU Yi-Shan1,2,3and LIU Ying1,2
(1. Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China; 2. Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524025, China; 3. Guangdong Key Laboratory of Control for Disease of Aquatic Economic Animals, Zhanjiang 524025, China)

Glutathione-S-transferase (GST, EC2.5.1.18) is the key antioxidant enzyme that catalyzes the conjugation of glutathione to several electrophilic substrates in living beings. Chlamydomonas sp. ICE-L is a rare ice algae living in the Antarctica where cold conditions and strong ultraviolet radiation are present all year round. In order to find out the role that GST in Chlamydomonas sp. ICE-L plays in acclimatizing to freezing polar environment, the expression of GST gene in Chlamydomonas sp. ICE-L was analyzed using real-time PCR under different temperatures. To evaluate the amount of template RNA in each real-time PCR reaction, gene fragments of β-actin was also amplified. The results showed that the Chlamydomonas sp. ICE-L GST gene could be expressed under all experimental temperatures. In the control group, as the temperature was 8℃, the accumulation of GST mRNA was maintained at identical level during 72h. At 0℃, GST mRNA accumulation decreased in the first 6h, which was followed by an increase and peaked at 36h (P<0.01) about more than twice of that in the control group, in the rest time the GST mRNA accumulation recovered to the level of control. At 14℃, the accumulation of GST mRNA was lower than the control group and slowly decreased during the entire experimental period and reached a quarter of the level in the control group at 72h. In addition, the pET-28a(+)-GST prokaryocyte expression vector was constructed and then transformed into E. coli BL21(DE3) to express the GST protein. The optimal expression conditions of pET-28a(+)-GST in E. coli BL21 included by 0.2 mmol/L concentration of IPTG, 37℃ and induction for 4h, and the product was mainly in the form of inclusion body. Using the HisTrap HP Columns, the expression product was separated and purified. Then the product was analyzed and confirmed by SDS-PAGE and Western blot, both results showed that the expression products with the molecular weight of 26 kD in the E. coli BL21(DE3) was the GST protein encoded by the GST gene from Chlamydomonas sp. ICE-L. At last, the E. coli BL21 containing the recombination plasmid (pET-28a(+)-GST) was treated with low temperature before growing on the agarose plate. As the recombination plasmid expressed mainly in the form of non-active aggregated monomers in E. coli BL21 induced with IPTG, we slowed the revolving speed and extended the induction time to express the GST protein with enzyme activation. The results of recombination plasmid treated with low temperature showed that the survival level of E. coli BL21 containing recombination plasmid (pET-28a(+)-GST) was higher than that of E. coli BL21 without this recombination plasmid in the first 4 days and reached the normal level at the 5thand 6thdays. Our results revealed that Chlamydomonas sp. ICE-L GST expressed in E. coli BL21 could improve the tolerance of E. coli to cold conditions, suggesting that GST may play an important role in the defense against freeze in the Chlamydomonas sp. ICE-L in Antarctica. These results provided valuable information on further investigation of the molecular mechanism of Chlamydomonas sp. ICE-L GST gene.

Chlamydomonas sp. ICE-L; Glutathione-S-transferase; Expression; Low temperature

Q786

A

1000-3207(2013)01-0016-06

10.7541/2013.16

2011-12-27;

2012-11-11

國(guó)家自然科學(xué)基金項(xiàng)目(40876102); 廣東省自然科學(xué)基金項(xiàng)目(S2011010005885)資助

彭躍躍(1985—), 男, 湖南人; 碩士研究生; 研究方向?yàn)楹Ｑ笪⑸飳W(xué)與病害防治。E-mail: pengyueyue8579@163.com

丁燏(1971—), 博士, 教授; 主要研究方向?yàn)楹Ｑ笪⑸飳W(xué)與病害防治。E-mail: dingy@gdou.edu.cn