過表達(dá)OsMPK17激酶蛋白質(zhì)增強(qiáng)了水稻的耐旱性

馬金姣 蘭金蘋,2 張 彤 陳 悅 郭亞璐,3 劉玉晴 燕高偉 魏 健 竇世娟 楊 明 李莉云 劉國振,*

過表達(dá)OsMPK17激酶蛋白質(zhì)增強(qiáng)了水稻的耐旱性

馬金姣1蘭金蘋1,2張 彤1陳 悅1郭亞璐1,3劉玉晴1燕高偉1魏 健1竇世娟1楊 明1李莉云1劉國振1,*

1河北農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院, 河北保定 071001;2河北北方學(xué)院生命科學(xué)研究中心, 河北張家口 075000;3中國農(nóng)業(yè)科學(xué)院農(nóng)業(yè)基因組研究所, 廣東深圳 518116

促分裂原活化蛋白質(zhì)激酶(mitogen-activated protein kinase, MAPK)在真核生物中高度保守, 在水稻逆境應(yīng)答反應(yīng)中也發(fā)揮著重要作用。本研究表達(dá)純化了水稻OsMPK17蛋白質(zhì), 制備了特異性抗體, 對(duì)多種非生物逆境脅迫下的蛋白質(zhì)樣品進(jìn)行免疫印跡分析, 發(fā)現(xiàn)OsMPK17蛋白質(zhì)在干旱脅迫下誘導(dǎo)表達(dá), 提示該蛋白質(zhì)在干旱脅迫應(yīng)答中發(fā)揮作用。對(duì)脫落酸和茉莉酸甲酯處理的離體葉片蛋白質(zhì)分析發(fā)現(xiàn), OsMPK17蛋白質(zhì)表達(dá)豐度下降, 提示該蛋白質(zhì)的功能發(fā)揮可能受激素調(diào)控。為此, 構(gòu)建了過表達(dá)OsMPK17蛋白質(zhì)的載體, 轉(zhuǎn)化水稻后篩選獲得了OsMPK17蛋白質(zhì)過表達(dá)的純合株系。田間種植鑒定結(jié)果表明, 轉(zhuǎn)基因株系的株高變矮、穗長變短、結(jié)實(shí)率降低。種子萌發(fā)期擬旱(PEG-6000)處理?xiàng)l件下, 過表達(dá)OsMPK17株系的種子長勢明顯比野生型好, 根長與芽長均顯著大于野生型。幼苗期失水試驗(yàn)表明, 轉(zhuǎn)基因植株的失水率低于野生型。在土培干旱脅迫后恢復(fù)澆水的試驗(yàn)中, 過表達(dá)OsMPK17蛋白質(zhì)的轉(zhuǎn)基因水稻生長也好于野生型。綜上, 過表達(dá)OsMPK17蛋白質(zhì)提高了水稻的耐旱性。本研究增進(jìn)了對(duì)水稻基因功能的了解。

水稻; MAPK蛋白質(zhì); 免疫印跡; 逆境脅迫; 過表達(dá); 基于抗體的蛋白質(zhì)組學(xué)

蛋白質(zhì)激酶可以使特定蛋白質(zhì)側(cè)鏈中的絲氨酸、蘇氨酸或酪氨酸殘基共價(jià)磷酸化[1], 它們在真核生物(酵母、哺乳動(dòng)物、人及植物)中廣泛存在[2-3]。促分裂原活化蛋白質(zhì)激酶(mitogen-activated protein kinase, MAPK)屬于絲/蘇氨酸蛋白質(zhì)激酶大家族, 通常由11個(gè)保守的亞結(jié)構(gòu)域組成[4]。真核生物中的激酶級(jí)聯(lián)反應(yīng)一般通過3個(gè)磷酸激酶(MAP3K、MAP2K和MAPK)依次使底物蛋白磷酸化轉(zhuǎn)導(dǎo)和放大信號(hào), 調(diào)控特定基因的表達(dá)[5]。MAPK處于這個(gè)級(jí)聯(lián)反應(yīng)的末端, 它被上游MAP2K活化后再磷酸化下游的元件, 如轉(zhuǎn)錄因子WRKY、MYB等, 將信號(hào)傳遞下去。植物MAPK級(jí)聯(lián)途徑在多種信號(hào)傳導(dǎo)過程中發(fā)揮著承上啟下的作用, 與許多生物及非生物脅迫反應(yīng)、激素反應(yīng)、細(xì)胞分化和發(fā)育過程相互關(guān)聯(lián), 形成調(diào)控植物正常生長和逆境應(yīng)答的網(wǎng)絡(luò)。

是第一個(gè)被鑒定的水稻MAPK基因, 受稻瘟病菌()侵染和機(jī)械損傷誘導(dǎo)表達(dá)[6]。和受脫落酸(Abscisic acid, ABA)、高鹽、干旱、高溫(37°C)或重金屬等非生物脅迫的誘導(dǎo)表達(dá), 而紫外線和低溫(12°C)不影響的表達(dá)水平, 但高溫(37°C)會(huì)降低OsWJUMK1的水平[7]。Shi等[8]證明在脫落酸信號(hào)途徑中能調(diào)高抗氧化物酶的活性。Xie等[9]證明OsMPK3能被OsMKK6磷酸化, 二者互作共同介導(dǎo)低溫信號(hào)的傳導(dǎo)[10]。和也參與鹽脅迫反應(yīng)。低溫脅迫會(huì)啟動(dòng)信號(hào)途徑, 提高水稻耐寒性[11]。OsMPK4與OsMKK1二者互作協(xié)調(diào)控制下游轉(zhuǎn)錄因子的表達(dá)并介導(dǎo)鹽脅迫應(yīng)答[12]。水稻在褐飛虱抗性反應(yīng)中發(fā)揮作用, 其證據(jù)有OsMPK5與褐飛虱抗性基因Bphi008a互作, 且OsMPK5可磷酸化Bphi008a, 在褐飛虱感染后OsMPK5的表達(dá)水平發(fā)生變化[13]。OsMAPK6參與OsMKKK10-OsMKK4信號(hào)通路, 對(duì)水稻的籽粒大小和重量發(fā)揮調(diào)節(jié)作用[14]。和等參與低溫脅迫應(yīng)答反應(yīng), 高溫和脫落酸處理還能使的表達(dá)上調(diào), 干旱和光照也對(duì)其有不同程度的影響[15]?；蛱岣叻N子萌發(fā)期對(duì)干旱和鹽脅迫的耐受性[16]。Lee等[17]發(fā)現(xiàn)在水稻鹽脅迫中起負(fù)調(diào)控作用。綜上可見, 多個(gè)水稻MAPK都在逆境脅迫應(yīng)答中發(fā)揮作用。所以進(jìn)一步鑒定逆境應(yīng)答相關(guān)的MAPK基因, 探討水稻逆境脅迫應(yīng)答機(jī)制具有重要的理論意義和應(yīng)用價(jià)值。

目前對(duì)水稻MAPK家族基因功能研究的線索主要來自遺傳分析及轉(zhuǎn)錄數(shù)據(jù), 其中基因在逆境脅迫反應(yīng)中的功能研究尚未見報(bào)道。本研究對(duì)不同逆境脅迫下的蛋白質(zhì)樣品進(jìn)行免疫印跡分析, 篩查發(fā)現(xiàn)OsMPK17蛋白質(zhì)在干旱脅迫下被誘導(dǎo)表達(dá), 提示該蛋白質(zhì)在干旱脅迫應(yīng)答中有作用。為了調(diào)查OsMPK17的功能, 構(gòu)建了過表達(dá)OsMPK17蛋白質(zhì)的載體, 轉(zhuǎn)化水稻后篩選獲得了OsMPK17蛋白質(zhì)過表達(dá)的純合株系, 在田間表型鑒定的基礎(chǔ)上, 通過PEG-6000擬旱處理、幼苗期失水試驗(yàn)和實(shí)際土培干旱脅迫試驗(yàn), 證明過表達(dá)OsMPK17蛋白質(zhì)的轉(zhuǎn)基因水稻生長優(yōu)于野生型, 該證據(jù)表明OsMPK17蛋白質(zhì)在干旱脅迫應(yīng)答過程中發(fā)揮作用。

1 材料與方法

1.1 供試材料

所用水稻品種為TP309 (粳稻)?？寺∮玫拇竽c桿菌菌株為DH5α, 融合蛋白質(zhì)表達(dá)菌株為BL21(DE3) pLysS和Codon Plus。原核表達(dá)載體為pET30a和pGST (由pGEM載體改造[18])。pUC57-3HA質(zhì)粒的插入片段由南京金斯瑞生物科技有限公司合成; pEASY-T1質(zhì)粒購自生工生物工程(上海)股份有限公司; pUBI-C4300質(zhì)粒由Pamela Ronald博士(UC Davis, USA)贈(zèng)送。

1.2 水稻OsMPK17基因的克隆

水稻基因全長cDNA質(zhì)粒AK070644 (Os05g50120)購自日本農(nóng)業(yè)生物資源研究所水稻基因組資源中心(Rice Genome Resource Center, National Institute of Agrobiological Sciences)。利用Primer CE軟件[19]設(shè)計(jì)引物, 由北京華大基因研究中心有限公司合成。上游引物序列為5'-GCATGGGCGGCCGCGCCCGCTC-3', 其中下畫線為I限制性內(nèi)切酶位點(diǎn), 下游引物序列為5'-GCGGTTTTCAGTTGAGCAAC-3', 其中下畫線為I限制性內(nèi)切酶位點(diǎn)。以帶有全長的cDNA的質(zhì)粒為模板進(jìn)行PCR擴(kuò)增, 對(duì)PCR產(chǎn)物和pET30a載體進(jìn)行雙酶切并切膠回收, 將連接后的產(chǎn)物轉(zhuǎn)化到大腸桿菌 DH5α中, 提取重組質(zhì)粒 DNA, 雙酶切驗(yàn)證后送北京華大基因研究中心有限公司測序驗(yàn)證。

1.3 融合蛋白質(zhì)表達(dá)純化

將測序確認(rèn)正確的pET30a-MPK17質(zhì)粒轉(zhuǎn)入表達(dá)菌Codon plus, 挑取單菌落過夜培養(yǎng), 按1∶10000的比例轉(zhuǎn)接至100 mL含50 μg mL–1卡那霉素的LB液體培養(yǎng)基中誘導(dǎo)表達(dá), 37°C振蕩培養(yǎng)至OD600為0.6~0.8, 按1∶200比例加入100 mmol L–1的IPTG, 25°C過夜培養(yǎng), 收菌后用20 mL 10 mmol L–1Tris-HCl (pH 8.0)將菌液懸浮, 超聲破碎(500 W, 60次, 每次10 s, 間隔15 s), 用His-tag beads進(jìn)行蛋白質(zhì)純化, 10% SDS-PAGE分離, 考染檢測重組蛋白質(zhì)的濃度和純度。

1.4 抗體制備

用大腸桿菌表達(dá)并純化后的OsMPK17蛋白質(zhì)做免疫原, 免疫小鼠制備單克隆抗體, 抗體的制備由北京華大蛋白質(zhì)研發(fā)中心有限公司完成。

1.5 水稻總蛋白質(zhì)提取及免疫印跡(Western blot, WB)檢測

將水稻樣品裝入離心管, 用液氮速凍, 在研磨機(jī)(鼎昊源科技, 型號(hào)TL2010)上振蕩, 1400 r min–1, 30 s, 低溫研磨直至粉末狀。按照樣品和蛋白質(zhì)提取緩沖液(62.5 mmol L–1Tris-HCl pH 7.4、10%甘油、2% SDS、1 mmol L–1PMSF、2 mmol L–1EDTA、5% β-巰基乙醇) 3∶8比例, 充分混勻后置冰上, 放置10 min, 每隔2 min 劇烈振蕩30 s, 12,000 ×, 4°C離心30 min后取上清液即為水稻組織總蛋白質(zhì)。經(jīng)SDS-PAGE進(jìn)行分離, 上樣體積一般為10 μL, 電泳完成后將蛋白質(zhì)轉(zhuǎn)移到PVDF膜上, 按文獻(xiàn)[20]描述的過程進(jìn)行WB檢測, 并用抗HSP抗體的檢測信號(hào)作為標(biāo)定上樣量的內(nèi)參[21], 二抗為HRP標(biāo)記的羊抗鼠二抗(北京華大蛋白質(zhì)研發(fā)中心有限公司)。用化學(xué)發(fā)光成像儀(MiniChemi 610, 北京賽智創(chuàng)業(yè)科技有限公司)檢測, Image J軟件[22]采集信號(hào)。

1.6 水稻非生物逆境脅迫處理

冷、熱、淹和恒光、恒暗等脅迫處理方法及部分樣品來自本實(shí)驗(yàn)室積累的蛋白質(zhì)樣品資源庫RiceS-A300[23]。

1.7 水稻離體葉片激素處理

參照文獻(xiàn)[24]進(jìn)行水稻離體葉片的激素處理。取4~6周齡的水稻幼苗, 剪取長度為2 cm的葉片, 置培養(yǎng)皿中, 加15 mL 100 μmol L–1脫落酸(abscisic acid, ABA)或100 μmol L–1茉莉酸甲酯(methyljasmonic acid, MeJA), 以滅菌水為對(duì)照, 在培養(yǎng)箱中培養(yǎng), 溫度為30°C, 光照周期為L12 h/D12 h, 取材時(shí)間點(diǎn)分別為0、6 h、12 h、1 d、2 d、3 d、4 d、5 d和6 d。

1.8 過表達(dá)轉(zhuǎn)基因載體構(gòu)建

為了便于后續(xù)檢測, 以pUC57為骨架, 通過基因合成改造成帶3HA和終止子的質(zhì)粒pUC57-3HA, 在3HA序列上游引入I和I限制性內(nèi)切酶位點(diǎn), 3HA之前有I酶切位點(diǎn), 之后是終止密碼子, 下游是I和d III限制性內(nèi)切酶位點(diǎn)。利用pEASY-T1質(zhì)粒構(gòu)建一個(gè)中間載體, 將pUC57-3HA中的I與d III雙酶切片段連接到同樣雙酶切的pEASY-T1質(zhì)粒中, 稱為pEASY-T1-3HA。將基因的擴(kuò)增產(chǎn)物用I和I雙酶切, 插入pEASY-T1-3HA, 測序驗(yàn)證后將含有目的基因和3HA的片段用I和dIII連接到pUBI-C4300質(zhì)粒中[25]。

1.9 水稻轉(zhuǎn)化

通過農(nóng)桿菌介導(dǎo)法將質(zhì)粒DNA轉(zhuǎn)入水稻, 在培養(yǎng)基中加甘露糖進(jìn)行篩選, 遺傳轉(zhuǎn)化由武漢伯遠(yuǎn)生物科技有限公司完成[26-27]。水稻遺傳轉(zhuǎn)化的主要步驟為, 從成熟胚誘導(dǎo)愈傷組織, 愈傷組織與根癌農(nóng)桿菌的共培養(yǎng), 抗性愈傷組織篩選, 預(yù)分化、分化、生根培養(yǎng)基培養(yǎng)出苗, 煉苗后溫室培養(yǎng)[28]。

1.10 水稻栽培及表型鑒定

將野生型和轉(zhuǎn)基因水稻種植于河北農(nóng)業(yè)大學(xué)西校區(qū)稻竹園, 在成熟期照相并測量農(nóng)藝性狀。測量5株以上株高、稻穗長度、結(jié)實(shí)率和分蘗數(shù), 計(jì)算平均值和方差。

1.11 水稻種子萌發(fā)期PEG-6000擬旱處理

參照文獻(xiàn)[29]進(jìn)行PEG-6000處理: 分別取對(duì)照和轉(zhuǎn)基因材料30粒種子, 用70%無水乙醇浸泡(搖床上輕輕搖晃) 5 min, 用滅菌水洗2次, 每次1 min, 用25%次氯酸鈉浸泡30 min, 用滅菌水清洗3次。在裝有2層濾紙的玻璃培養(yǎng)皿中倒入15 mL 20% PEG-6000, 以滅菌水為對(duì)照, 將消毒后的種子用鑷子均勻擺放在培養(yǎng)皿中, 培養(yǎng)箱溫度為30°C, 光周期L12 h/D12 h, 7 d后測量株高和芽長等, 并照相記錄。

1.12 水稻幼苗失水率調(diào)查

取溫室培養(yǎng)的四葉期水稻幼苗, 剪取長度為3 cm左右的葉片, 置室溫(30°C)條件下, 每0.5 h稱重一次。以0時(shí)間點(diǎn)重量為0, 指定時(shí)間點(diǎn)為W, 失水率(%) = (0?W) × 100/W。試驗(yàn)重復(fù)3次, 計(jì)算平均值和方差[30-31]。

1.13 幼苗期土培干旱處理

盆栽參試水稻材料, 種子經(jīng)30°C浸泡3 d露白, 播于蛭石土(土壤與蛭石1∶1), 30°C、光周期L12 h/D12 h培養(yǎng)5 d, 停止?jié)菜? 讓花盆自然干燥, 經(jīng)一定時(shí)間的干旱脅迫后, 植株出現(xiàn)萎蔫伴有死亡的跡象時(shí), 恢復(fù)澆水, 觀察并照相記錄水稻狀況。

2 結(jié)果與分析

2.1 OsMPK17融合蛋白質(zhì)的表達(dá)及抗體制備

利用設(shè)計(jì)的PCR引物, 以帶有全長OsMPK17 cDNA的質(zhì)粒為模板進(jìn)行擴(kuò)增(圖1-A), 擴(kuò)增產(chǎn)物為1800 bp左右, 符合預(yù)期, 將PCR產(chǎn)物和pET30a表達(dá)載體進(jìn)行雙酶切, 連接轉(zhuǎn)化細(xì)菌后提取質(zhì)粒DNA進(jìn)行雙酶切驗(yàn)證(圖1-B), 酶切產(chǎn)物為2條帶, 分子量較高的是載體帶, 約為6000 bp, 分子量較低的是插入片段, 約為1800 bp。將雙酶切正確的質(zhì)粒再進(jìn)行測序驗(yàn)證, 挑取序列正確的重組質(zhì)粒pET30a- MPK17轉(zhuǎn)入表達(dá)菌Codon plus中, 挑取單菌落進(jìn)行誘導(dǎo)表達(dá)。收集的菌體經(jīng)超聲破碎后離心分離, 用His-tag beads親和層析柱純化, 蛋白質(zhì)樣品經(jīng)SDS-PAGE分離后, 考染觀察(圖1-C), 在大腸桿菌的沉淀中能清楚地看到誘導(dǎo)條帶。利用純化后的融合OsMPK17蛋白質(zhì)作為免疫原, 免疫小鼠制備單克隆抗體。

2.2 苗期非生物逆境脅迫下OsMPK17蛋白質(zhì)的表達(dá)特征

本實(shí)驗(yàn)室張劍碩等[23]報(bào)道了水稻RiceS-A300資源庫構(gòu)建的工作, 為了獲得OsMPK17在逆境脅迫應(yīng)答過程中可能的功能線索, 利用所制備的特異性抗體, 對(duì)這些樣品進(jìn)行了WB分析, 發(fā)現(xiàn)在干旱脅迫過程中, OsMPK17的表達(dá)量持續(xù)升高, 而對(duì)照樣品中OsMPK17蛋白質(zhì)的表達(dá)量維持穩(wěn)定(圖2-A), 提示該基因在干旱脅迫反應(yīng)中可能發(fā)揮作用。對(duì)其他脅迫處理的材料進(jìn)行WB分析, 沒有檢測到明顯的表達(dá)變化(數(shù)據(jù)未附)。接下來, 以離體水稻葉片為材料, 進(jìn)行ABA和MeJA處理, 提取蛋白質(zhì)后進(jìn)行WB分析, 發(fā)現(xiàn)ABA和MeJA處理均能降低OsMPK17蛋白質(zhì)的豐度(圖2-B), 比較而言, ABA處理的效果更為明顯, 由此推測OsMPK17蛋白質(zhì)的功能發(fā)揮與ABA、MeJA等激素有一定相關(guān)性。

圖1 水稻OsMPK17基因的克隆與融合蛋白質(zhì)表達(dá)

A:水稻基因的PCR擴(kuò)增: 以帶有全長序列的質(zhì)粒DNA為模板, 用上游引物5′-GCGGTACCATGGG CGGCCGCGCCCGCTC-3′, 下游引物5′-GCGAGCTCGGTTTTC AGTTGAGCAAC-3′, 擴(kuò)增s基因。B: pET30a-MPK17重組質(zhì)粒的雙酶切驗(yàn)證: 將PCR產(chǎn)物與pET30a用I+I雙酶切, 連接后轉(zhuǎn)化克隆菌DH5α, 提取質(zhì)粒后再進(jìn)行雙酶切鑒定。C:融合蛋白質(zhì)OsMPK17的誘導(dǎo)表達(dá)及考染檢測: 取pET30a-MPK17酶切驗(yàn)證的質(zhì)粒進(jìn)行測序再驗(yàn)證, 將測序正確的質(zhì)粒轉(zhuǎn)化表達(dá)菌Codon plus誘導(dǎo)表達(dá)。在含50 μg mL–1卡那霉素的LB液體培養(yǎng)基中誘導(dǎo)表達(dá), 振蕩培養(yǎng)至OD600為0.6~0.8, 加入IPTG, 25°C過夜培養(yǎng), 收菌后超聲破碎, 離心取上清液(S)和沉淀(P), 0: 0時(shí)間點(diǎn)培養(yǎng)物, 用10% SDS-PAGE分離、考染。M為分子量標(biāo)記; PCR為擴(kuò)增產(chǎn)物; K+S為I+I雙酶切產(chǎn)物。

A: PCR amplification of ricegene. A plasmid containing full-lengthgene was used as template for PCR amplification ofgene using primers 5′-GCGGTACCATGGG CGGCCGCGCCCGCTC-3′ and 5′-GCGAGCTCGGTTTTCAGTT GAGCAAC-3′. B: Verification of recombinant pET30a-MPK17 plasmid by double digestion usingI andI. The PCR products and pET30a plasmid DNA were digested byI andI, the ligation product was used to transform DH5α. Recombinant plasmid was verified by double digestion. C: Induction of fusion protein OsMPK17 and Coomassie blue staining. Correct pET30a- MPK17 plasmid verified by double digestion was double checked by sequencing. Sequencing verified plasmid was transformed to Codon plus bacterial strain to express fusion protein. The bacteria was cultured in LB medium containing 50 μg mL–1kanamycin and IPTG which was added when the OD600reached 0.6–0.8. The bacteria was collected after over night culture at 25°C and disrupted by sonication. The supernatant (S) and pellet (P) were obtained after centrifugation and total protein was separated by 10% SDS-PAGE and stained with Coomassie blue. 0: Total protein isolated at 0 time point. M: Molecular weight marker; PCR: Amplification products; K+S: Double digestion product usingI andI.

2.3 過表達(dá)OsMPK17轉(zhuǎn)基因載體的構(gòu)建

設(shè)計(jì)PCR引物, 以cDNA質(zhì)粒為模板, 擴(kuò)增獲得了全長的基因(圖3-A), 雙酶切后裝入pEASY-3HA中間載體, 測序驗(yàn)證后, 用內(nèi)切酶d III和I切下片段, 電泳檢測到1848 bp的插入片段(圖3-B), 裝入水稻轉(zhuǎn)化質(zhì)粒pUBI-C4300中, 獲得了pUBI-C4300-MPK17轉(zhuǎn)化載體, 經(jīng)I和I酶切檢測到符合預(yù)期的插入片段(圖3-C), 證明獲得了正確的過表達(dá)載體。

圖2 水稻OsMPK17蛋白質(zhì)的表達(dá)特征分析

A: 干旱脅迫: 發(fā)芽后生長5 d的水稻幼苗在20% PEG-6000中水培, 分別于脅迫處理的0、1 h、2 h、4 h、8 h、12 h、1 d 、2 d和3 d取地上部樣品, 提取總蛋白質(zhì)后進(jìn)行免疫印跡(Western blot, WB)分析。CK: 對(duì)照; Drought: PEG-6000脅迫處理。HSP: 以HSP82抗體檢測的信號(hào)作為上樣內(nèi)參。B: 激素處理: 剪下水稻幼苗葉片在培養(yǎng)皿中培養(yǎng), 分別添加100 μmol L–1ABA或100 μmol L–1MeJA進(jìn)行激素處理, 取材時(shí)間點(diǎn)為0、6 h、12 h、1 d、2 d、3 d、4 d、5 d和6 d, 以水培為CK, 提取葉片樣品總蛋白質(zhì)進(jìn)行WB分析。HSP: 以HSP82抗體檢測的信號(hào)作為上樣內(nèi)參。

A: Drought stress treatment: rice seedlings grown for 5 days were treated by 20% PEG-6000. Leaf samples were collected at 0, 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, and 3 d respectively; WB analysis were carried out for isolated total proteins. HSP: Loading control for WB analysis using HSP82 antibody. B: Treatment with hormones: leaves of rice were cultured in petri dish, 100 μmol L–1ABA or 100 μmol L–1MeJA was supplemented as hormone treatments. Samples were collected at 0, 6 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, and 6 d time points, respectively. Total proteins were isolated and analyzed by WB. HSP: Loading control for WB analysis using HSP82 antibody.

2.4 水稻轉(zhuǎn)化及陽性植株的鑒定

以水稻品種TP309為受體, 通過農(nóng)桿菌介導(dǎo)的途徑轉(zhuǎn)化水稻, T0代獲得了11個(gè)轉(zhuǎn)基因株系, PCR鑒定其中10個(gè)為陽性, 收獲陽性植株種子, T1代再次鑒定篩選陽性株系, 收獲種子后T2代獲得了純合的過表達(dá)OsMPK17的水稻材料, 4個(gè)轉(zhuǎn)基因株系(A202、A204、A210和A212)的PCR和WB檢測結(jié)果如圖4所示, 所檢測的4個(gè)轉(zhuǎn)基因株系的植株全部為PCR陽性和WB陽性, 而野生型受體水稻均表現(xiàn)陰性。由于過表達(dá)的OsMPK17蛋白質(zhì)帶有3×HA標(biāo)簽, 分子量比水稻中原有的OsMPK17蛋白質(zhì)稍大, 所以在WB中可以檢測到2個(gè)條帶, 分子量稍大的是超表達(dá)的版本, 記做MPK17-OX, 分子量較小的是水稻中原來的版本, 記作MPK17-Native, 比較2個(gè)條帶的信號(hào)強(qiáng)度可見, 二者的豐度比較接近。

2.5 過表達(dá)OsMPK17蛋白質(zhì)轉(zhuǎn)基因水稻植株的表型鑒定

在大田栽培生長過程中調(diào)查了水稻植株的表型和主要農(nóng)藝性狀, 從圖5可見, 過表達(dá)OsMPK17蛋白質(zhì)轉(zhuǎn)基因植株的株高、穗長和結(jié)實(shí)率等指標(biāo)均低于野生型, 而分蘗數(shù)的差別不明顯, 說明OsMPK17過表達(dá)對(duì)水稻的正常生長產(chǎn)生了不利的影響。

2.6 過表達(dá)OsMPK17水稻植株發(fā)芽期耐旱性鑒定

在干旱脅迫過程中, OsMPK17蛋白質(zhì)的表達(dá)豐度提高, 提示該蛋白質(zhì)在抵抗干旱脅迫過程中發(fā)揮正調(diào)控作用。圖6-A表明4個(gè)過表達(dá)OsMPK17的株系均比野生型生長健壯, 其根長和芽長(圖6-B)均明顯高于野生型對(duì)照, 說明OsMPK17蛋白質(zhì)的過表達(dá)提高了水稻發(fā)芽期的耐旱性。

2.7 過表達(dá)OsMPK17水稻植株幼苗期的失水率鑒定

為了比較過表達(dá)OsMPK17蛋白質(zhì)水稻的耐旱性, 進(jìn)一步調(diào)查了水稻植株在苗期的失水率。圖7表明4個(gè)過表達(dá)株系的失水率均低于或不高于野生型對(duì)照, 失水率試驗(yàn)支持過表達(dá)OsMPK17植株具有較好的保水性, 這可能是其耐旱性提高的原因之一。

2.8 過表達(dá)OsMPK17水稻植株苗期耐旱性鑒定

為了進(jìn)一步鑒定轉(zhuǎn)基因水稻的耐旱性, 將2個(gè)轉(zhuǎn)基因株系(A202和A212)與野生型對(duì)照進(jìn)行同盆栽培, 以便使培養(yǎng)條件盡量保持一致, 種子發(fā)芽后正常盆栽培養(yǎng)5 d, 停止?jié)菜? 花盆土壤自然干燥, 使水稻幼苗承受實(shí)際的干旱脅迫, 經(jīng)不同天數(shù)的干旱脅迫后, 待部分植株出現(xiàn)萎蔫并呈現(xiàn)葉片有50%干枯時(shí), 恢復(fù)澆水并觀察水稻苗的生長狀況(圖8)。轉(zhuǎn)基因苗(右側(cè))在脅迫處理時(shí)和恢復(fù)澆水后都表現(xiàn)比野生型水稻苗(左側(cè))有更多的綠色部分, 說明轉(zhuǎn)基因苗具有較強(qiáng)的耐旱性。

圖3 水稻OsMPK17基因過表達(dá)載體的構(gòu)建與鑒定

A: 水稻基因的PCR擴(kuò)增; B:d III +I雙酶切鑒定pEASY-MPK17-3HA重組質(zhì)粒; C:I+I雙酶切鑒定pUBI-C4300-MPK17重組質(zhì)粒。以帶有目的基因cDNA的質(zhì)粒為模板, 用上游引物5′-GCATGGGCGGCCGCGCCC GCTC-3′ (下畫線為I限制性內(nèi)切酶位點(diǎn))和下游引物5′-GCGGTTTTCAGTTGAGCAAC-3′ (下畫線為I限制性內(nèi)切酶位點(diǎn))進(jìn)行PCR擴(kuò)增。將擴(kuò)增的片段插入中間載體pEASYT1-3HA, 雙酶切驗(yàn)證。將測序正確的中間載體切膠回收目的片段, 再用I+I雙酶切插入轉(zhuǎn)化載體pUBI-C4300, 獲得重組質(zhì)粒DNA雙酶切驗(yàn)證。M: 分子量標(biāo)記; PCR: 擴(kuò)增產(chǎn)物; H+X:d III+I雙酶切; K+S:I+I雙酶切。

A: PCR amplification of ricegene; B:d III+I restriction enzyme digestion of recombinant pEASY-MPK17-3HA plasmid; C:I+I restriction enzyme digestion of recombinant pUBI-C4300-MPK17 plasmid. PCR amplification ofgene using plasmid containing full-lengthcDNA as template, the primers used were 5′-GCATGGGC GGCCGCGCCCGCTC-3′ (I restriction site was underlined) and 5′-GCGGTTTTCAGTTGAGCAAC-3′ (I restriction site was underlined). The amplified fragment was inserted into pEASY-3HA vector and verified by double digestion. Sequence verified pEASY-MPK17-3HA was digested byI+I, the fragment was inserted into pUBI-C4300 and verified by double digestion. M: Molecular weight marker; PCR: PCR amplification product; H+X:d III+I restriction enzyme digestion; K+S:I+I restriction enzyme digestion.

3 討論

蛋白質(zhì)是生命活動(dòng)的主要執(zhí)行者, 蛋白質(zhì)表達(dá)特征中蘊(yùn)含著功能相關(guān)的線索。本研究制備了水稻OsMPK17蛋白質(zhì)特異的抗體, 經(jīng)WB分析發(fā)現(xiàn)該蛋白質(zhì)在干旱脅迫過程中表達(dá)上調(diào), 由此推測其在耐旱過程中發(fā)揮正調(diào)控作用, 為此構(gòu)建了OsMPK17的過表達(dá)載體, 采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化水稻, 獲得了過表達(dá)OsMPK17蛋白質(zhì)的轉(zhuǎn)基因株系, 在萌發(fā)期對(duì)轉(zhuǎn)基因材料進(jìn)行PEG-6000的擬旱處理、葉片失水率調(diào)查和幼苗期的實(shí)際干旱試驗(yàn), 結(jié)果均表明過表達(dá)OsMPK17轉(zhuǎn)基因植株提高了水稻的耐旱性。

在轉(zhuǎn)基因材料的鑒定方面, 本研究除采用常規(guī)的PCR技術(shù)鑒定陽性植株外, 還通過WB對(duì)OsMPK17蛋白質(zhì)的過表達(dá)進(jìn)行了鑒定, 鑒定結(jié)果表明絕大部分PCR陽性植株均表現(xiàn)WB陽性, 但也發(fā)現(xiàn)有少數(shù)PCR陽性的植株表現(xiàn)WB陰性(數(shù)據(jù)未附), 說明PCR陽性并不能確保蛋白質(zhì)的過表達(dá), 通過WB進(jìn)行鑒定是必要的。WB的檢測直接證明了過表達(dá)OsMPK17蛋白質(zhì)的存在。從技術(shù)上講, PCR具有更高的靈敏度, 但也容易出現(xiàn)假陽性, 而WB檢測的靈敏度較低, 一般不容易出現(xiàn)假陽性結(jié)果。有意思的是, 過表達(dá)的OsMPK17與野生型OsMPK17蛋白質(zhì)的分子量有所不同, 能夠被電泳區(qū)分, 所以WB可清晰地檢測到野生型和過表達(dá)的OsMPK17蛋白質(zhì)。根據(jù)條帶信號(hào)的強(qiáng)度, 可大致判斷出二者的豐度基本相近。用WB對(duì)轉(zhuǎn)基因材料進(jìn)行鑒定, 獲得了轉(zhuǎn)基因材料中目標(biāo)蛋白質(zhì)的有無、豐度及分子量等信息。

圖4 過表達(dá)OsMPK17蛋白質(zhì)轉(zhuǎn)基因水稻篩選與鑒定

上部: PCR結(jié)果; 中部: WB檢測轉(zhuǎn)基因水稻中OsMPK17蛋白質(zhì); 下部: HSP檢測的信號(hào)為上樣參照。WT: 野生型; A202、A204、A210和A 212為不同的轉(zhuǎn)基因株系; 1、2、3、4、5、6、7、8和9為同一株系內(nèi)不同的單株; PCR: PCR產(chǎn)物; MPK17-OX: 過表達(dá)的OsMPK17蛋白質(zhì); MPK17-Native: 水稻中原來的OsMPK17蛋白質(zhì)。

Upper panel: PCR product; Middle panel: WB detection of OsMPK17 protein in transgenic rice plants; Lower panel: HSP signal was used as loading control; WT: wildtype rice plants; A202, A204, A210, and A212 are independent transgenic lines; 1, 2, 3, 4, 5, 6, 7, 8, and 9 are independent plants among the same transgenic lines; PCR: PCR products; MPK17-OX: Over expressed OsMPK17 protein; MPK17-Native: the original form of OsMPK17 protein in rice.

圖5 過表達(dá)OsMPK17對(duì)水稻表型及農(nóng)藝性狀的影響

上部照片: 4個(gè)轉(zhuǎn)基因株系(A202、A204、A210和A212)及對(duì)照水稻成熟期整株和穗部照片; 下部柱狀圖: 4個(gè)轉(zhuǎn)基因株系及對(duì)照的株高、穗長、結(jié)實(shí)率和分蘗數(shù)的柱狀圖。每個(gè)指標(biāo)測量5個(gè)以上單株, 計(jì)算平均值和方差。

Photographs on the upper panel: rice whole plants and ears at mature stage of four transgenic lines and control. Bar graphs on the lower panel: plant height, spike length, seed setting rate, and tillers number of the four transgenic lines and control.

圖6 過表達(dá)OsMPK17蛋白質(zhì)水稻發(fā)芽期耐旱性鑒定

A: 水稻種子萌發(fā)照片。上部: 對(duì)照(水); 下部: 干旱脅迫(20% PEG-6000)。B: 水稻種子萌發(fā)的根長和芽長柱狀圖。WT為野生型; A202、A204、A210和A212為4個(gè)轉(zhuǎn)基因株系。試驗(yàn)重復(fù)3次, 計(jì)算平均值和方差。*表示在0.05水平差異顯著;**表示在0.01水平差異顯著。

A: Photographs for seeds at germination. Upper panel: control (H2O); Lower panel: drought stress (20% PEG-6000) treatment. B: Bar graphs of root and shoot lengths for seeds at germination. WT: wild type; A202, A204, A210, and A212 are transgenic lines. Experiments were carried out with three replicates; average and standard derivations were calculated.*Significant at< 0.05.**Significant at< 0.01.

利用前期建立的RiceS-A300水稻蛋白質(zhì)樣品資源庫[23], 比較方便地實(shí)現(xiàn)了OsMPK17蛋白質(zhì)在各種非生物逆境脅迫下表達(dá)特征的調(diào)查, 由此發(fā)現(xiàn)OsMPK17蛋白質(zhì)在干旱脅迫條件下表達(dá)豐度被明顯誘導(dǎo)上調(diào), 而在其他非生物逆境脅迫下沒有發(fā)現(xiàn)明顯的變化, 蛋白質(zhì)表達(dá)特征數(shù)據(jù)提示該蛋白質(zhì)可能在干旱脅迫下發(fā)揮作用。另外, 我們也檢測了轉(zhuǎn)基因水稻在干旱脅迫下OsMPK17的豐度變化, 未檢測到原有版本OsMPK17-Native條帶信號(hào)的明顯增強(qiáng), 這可能是由于過表達(dá)的版本OsMPK17-OX發(fā)揮作用, 抑制了OsMPK17-Native的誘導(dǎo)表達(dá)。通過對(duì)激素處理的離體葉片中OsMPK17蛋白質(zhì)分析, 發(fā)現(xiàn)ABA和MeJA處理會(huì)降低OsMPK17的表達(dá)豐度。

圖7 過表達(dá)OsMPK17蛋白質(zhì)植株的失水率鑒定

WT為野生型; A202、A204、A210和A212為4個(gè)轉(zhuǎn)基因株系。在水稻幼苗四葉期剪取3 cm左右葉片, 在室溫下(30°C), 每30 min稱重1次, 試驗(yàn)重復(fù)3次, 計(jì)算平均值和方差。

WT: wild type; A202, A204, A210, and A212 are transgenic lines. At four leaves stage, leaf blades were cut into pieces at about 3 cm, which were weighed every 30 min at room temperature (30°C). The experiment were repeated three times; the average and standard derivation were calculated.

圖8 過表達(dá)OsMPK17蛋白質(zhì)轉(zhuǎn)基因水稻苗期耐旱性鑒定

水稻苗期干旱脅迫及恢復(fù)澆水試驗(yàn), 照片分別為0時(shí)間點(diǎn)、干旱脅迫8 d和恢復(fù)澆水3 d時(shí)。WT為野生型, A202和A212為過表達(dá)OsMPK17的轉(zhuǎn)基因株系。

WT: wild type; A202 and A212 were transgenic lines overexpressed OsMPK17 protein. The drought and restore experiments were carried out at seedling stage; the photographs were taken at 0 time point, eight days after drought treatment, and re-watering for 3 days.

對(duì)不同發(fā)育時(shí)期、不同部位組織中OsMPK17蛋白質(zhì)的豐度分析發(fā)現(xiàn), OsMPK17主要在葉片中表達(dá), 在其他檢測的部位中, 包括根、莖、葉鞘、幼穗、花藥、穎殼等, 均沒有檢測到OsMPK17蛋白質(zhì)(數(shù)據(jù)未附), 由此推測其功能應(yīng)該主要通過葉片來發(fā)揮。對(duì)轉(zhuǎn)錄數(shù)據(jù)進(jìn)行挖掘分析(http://rice.plantbiology. msu.edu/)表明,基因的轉(zhuǎn)錄主要在花藥中, 在其他部位的轉(zhuǎn)錄信號(hào)很低(附表1)。比較蛋白質(zhì)和轉(zhuǎn)錄信息可以看出, 二者相關(guān)性不大。此種情況下, 基于WB分析獲得的蛋白質(zhì)豐度信息更為直觀, 也應(yīng)該更具參考價(jià)值。蛋白質(zhì)的表達(dá)特征既是功能表現(xiàn)的一種外在形式, 也是其功能的組成部分。開展重要蛋白質(zhì)的表達(dá)特征調(diào)查具有重要的意義, 這也是基于抗體的靶向蛋白質(zhì)組學(xué)策略試圖發(fā)揮作用的方向[32]。

水稻基因組中有17個(gè)MAPK基因, 目前, 已經(jīng)鑒定到多個(gè)MAPK在生物脅迫或非生物脅迫反應(yīng)中發(fā)揮功能。如通過RNAi技術(shù)降低OsMPK5表達(dá)后, 也降低了水稻對(duì)ABA的敏感性以及對(duì)干旱、鹽和冷脅迫的敏感性, 但提高了對(duì)稻瘟病菌和白葉枯病菌的抗性[10,33-34]?？梢哉J(rèn)為, OsMPK5在水稻的脅迫反應(yīng)中起著兩方面的作用, 既可正調(diào)控ABA途徑中的非生物脅迫反應(yīng), 又能負(fù)調(diào)控對(duì)稻瘟病和白葉枯病的抗病反應(yīng)。此外, OsMPK7能被多種生物脅迫和非生物脅迫誘導(dǎo)表達(dá)[10,15,35], OsMPK17-1 (OsBWMK1)和OsMPK4 (OsMPK2)也參與植物的防御反應(yīng)[6,36]。本研究中對(duì)OsMPK17的功能闡釋增進(jìn)了對(duì)水稻MAPK蛋白質(zhì)激酶的了解。本研究通過WB檢測到OsMPK17蛋白質(zhì)在激素ABA和MeJA處理后, 表達(dá)豐度下降, 由此可推測OsMPK17蛋白質(zhì)介導(dǎo)的水稻耐旱性可能與激素相關(guān)。據(jù)報(bào)道, 高水平的ABA含量會(huì)提高種子萌發(fā)期對(duì)旱的耐受性、抑制種子發(fā)芽、側(cè)根形成、幼苗生長等, 通過促進(jìn)氣孔的閉合減少水分蒸騰來調(diào)控旱脅迫應(yīng)答反應(yīng)[37-43]。此外, 外源ABA的處理會(huì)影響植物中許多MAPK基因的表達(dá), MAPK參與了ABA介導(dǎo)的多種信號(hào)通路, 包括氧化防御、保衛(wèi)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)和種子的萌發(fā)等[44-47]。在煙草和擬南芥中, MeJA和SA可以誘導(dǎo)MPK6和MPK3的表達(dá)及SIPK的瞬時(shí)表達(dá)[48-49]。這些數(shù)據(jù)提示MAPK的功能發(fā)揮與SA、MeJA及ABA等激素有密切的關(guān)系。一般認(rèn)為干旱能誘導(dǎo)ABA含量的增加, 本文研究結(jié)果表明干旱也能誘導(dǎo)OsMPK17蛋白質(zhì)的表達(dá), 并增強(qiáng)了水稻的耐旱性。據(jù)此可推測ABA含量增加和OsMPK17的豐度增加都是干旱應(yīng)答的正調(diào)控因子。但是本研究又發(fā)現(xiàn), ABA處理會(huì)下調(diào)OsMPK17的豐度, 所以二者負(fù)相關(guān)。根據(jù)這些數(shù)據(jù)可以推測, 在干旱脅迫下ABA的含量會(huì)被誘導(dǎo)提高, 水稻應(yīng)該表現(xiàn)耐旱性, 但ABA含量的提高能抑制OsMPK17蛋白質(zhì)的表達(dá), 如此又造成水稻的耐旱性下降, 這樣兩個(gè)因素在水稻中達(dá)到平衡, 其凈結(jié)果表現(xiàn)為正常情況下水稻的耐旱性只能維持在一個(gè)特定的水平。本研究結(jié)果鑒定到OsMPK17蛋白質(zhì)與干旱脅迫應(yīng)答的相關(guān)性, 可能是通過激素介導(dǎo)響應(yīng)的, 今后可在耐旱性增強(qiáng)機(jī)制、激素是如何發(fā)揮作用等方面開展進(jìn)一步的工作。

4 結(jié)論

表達(dá)了水稻OsMPK17蛋白質(zhì), 制備了特異抗體, OsMPK17蛋白質(zhì)在干旱脅迫條件下表達(dá)量升高, 在ABA和MeJA等激素處理下表達(dá)量下降。獲得了過表達(dá) OsMPK17蛋白質(zhì)的轉(zhuǎn)基因水稻植株, 其株高、穗長和結(jié)實(shí)率等農(nóng)藝指標(biāo)均低于對(duì)照。過表達(dá)OsMPK17蛋白質(zhì)提高了水稻發(fā)芽期的耐旱性, 降低了幼苗期的失水率, 實(shí)際干旱恢復(fù)試驗(yàn)也表明過表達(dá)OsMPK17植株具有較好的耐旱性。

附表1基因在不同組織中的轉(zhuǎn)錄豐度比較

Supplementary table 1 Transcriptional abundance comparison ofgene among different rice tissues

組織 LibrariesFPKM 四葉期幼苗 Seedling four-leaf stage0.640 幼苗地上部 Shoots0.904 20 d的葉片 20-day leaves0.637 抽穗前花序 Pre-emergence inflorescence3.036 抽穗后花序 Post-emergence inflorescence6.281 花藥 Anther169.643 雌蕊 Pistil2.371 開花后5 d種子 5 DAP seed5.971 開花后10 d種子 10 DAP seed10.372 開花后25 d幼胚 25 DAP embryo7.719 開花后25 d胚乳 25 DAP endosperm7.230

從數(shù)據(jù)庫http://rice.plantbiology.msu.edu/下載基因的轉(zhuǎn)錄信息, 列于表中。FPKM: 每百萬被測到的外元中每千堿基所含目標(biāo)片段數(shù)。DAP: 授粉后天數(shù)。

Transcriptomic abundance of ricegene was downloaded from http://rice.plantbiology.msu.edu/ database and listed in the table. FPKM: fragments per kilobase of exon per million fragments mapped. DAP: days after pollination.

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Overexpression of OsMPK17 protein enhances drought tolerance of rice

MA Jin-Jiao1, LAN Jin-Ping1,2, ZHANG Tong1, CHEN Yue1, GUO Ya-Lu1,3, LIU Yu-Qing1, YAN Gao-Wei1, WEI Jian1, DOU Shi-Juan1, YANG Ming1, LI Li-Yun1, and LIU Guo-Zhen1,*

1College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei, China;2Research Center for Life Sciences, Hebei North University, Zhangjiakou 075000, Hebei, China;3Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518116, Guangdong, China

Mitogen-activated protein kinase (MAPK) highly conserved in eukaryotes plays important roles in stress responses in plant. In this study, full-lengthgene was cloned and fusion protein was expressed. The purified protein was used as immunogen to generate monoclonal antibody. Western blot (WB) analyses were carried out for protein samples isolated from tissues under different abiotic stresses. The expression ofwas induced by drought stress, suggesting that the OsMPK17 protein may play a role in drought stress response. Proteins isolated from leaves treated with abscisic acid (ABA) or methylene jasminate acid (MeJA) demonstrated a decrease of OsMPK17 protein abundance, suggesting that hormones may be involved in the function of the protein. The overexpression vector of OsMPK17 protein was established and transformed into TP309 via-mediated protocol. Homozygous transgenic lines for overexpression of OsMPK17 protein were obtained. In the field planting experiment, the plant height and the spike length of transgenic lines shortened and the seed setting rate decreased. At seed germination stage, under the condition of PEG-6000 treatment, the seeds of overexpressed OsMPK17 protein lines grew better and the length of root and shoot was significantly longer than those of the wild type. At seedling stage, transgenic plants showed lower water loss rate when exposed in the air. The transgenic rice with overexpressed OsMPK17 protein grew better than the wild type in the experiment with soil drought stress and re-watering then. In conclusion, the overexpressed OsMPK17 protein enhances drought tolerance of rice. This study enhances the understanding for the function of OsMPK17 protein.

rice; MAPK protein; Western blot; stress; overexpression; antibody-based proteomics

2019-02-26;

2019-08-09;

2019-09-04.

10.3724/SP.J.1006.2020.92007

劉國振, E-mail: gzhliu@hebau.edu.cn

E-mail: majinjiao_mbb@126.com

本研究由國家自然科學(xué)基金項(xiàng)目(31171528)資助。

This study was supported by the National Natural Science Foundation of China (31171528).

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20190903.1630.006.html