萊茵衣藻type I metacaspase基因克隆及其參與調(diào)控程序性細(xì)胞死亡研究*

張恒宇 柳逸群 葛 源

萊茵衣藻type I metacaspase基因克隆及其參與調(diào)控程序性細(xì)胞死亡研究*

張恒宇1柳逸群2葛 源1①

(1. 中國海洋大學(xué)海洋生命學(xué)院 青島 266003；2. 中國海洋大學(xué)水產(chǎn)學(xué)院 青島 266003)

Metacaspase是在原生動物、真菌和植物中發(fā)現(xiàn)的一種具有底物精氨酸/賴氨酸特異性的半胱氨酸蛋白酶。根據(jù)蛋白質(zhì)結(jié)構(gòu)特征可將metcaspase分為typeⅠ與typeⅡ兩種類型，均參與調(diào)控多種植物與原生動物程序性細(xì)胞死亡。本研究根據(jù)GenBank數(shù)據(jù)庫萊茵衣藻() type I metacaspase基因(GenBank No. XM_001696904)序列，采用巢式PCR克隆獲取type I metcaspase基因開放閱讀框(ORF)序列并命名為CrMC1。CrMC1 ORF全長987 bp，推測編碼1個包含405個氨基酸的蛋白質(zhì)。通過與已知物種type I metacaspase氨基酸序列進(jìn)行同源序列比對發(fā)現(xiàn)，CrMC1具有高度保守的p20、p10、連接區(qū)結(jié)構(gòu)域以及精氨酸、半胱氨酸活性中心位點(diǎn)。研究顯示，在H2O2誘導(dǎo)的萊茵衣藻程序性細(xì)胞死亡過程中，CrMC1表達(dá)量顯著提高(<0.05)，2 h后達(dá)到峰值，3 h時下降至對照組同一水平。結(jié)果表明，萊茵衣藻type I metacaspase基因CrMC1參與H2O2誘導(dǎo)的萊茵衣藻程序性細(xì)胞死亡調(diào)控。

Metacaspase；萊茵衣藻；程序性細(xì)胞死亡

萊茵衣藻()是綠藻門(Chlorophyceae)、團(tuán)藻目(Volvocales)、衣藻屬()的一種單細(xì)胞真核微藻，具有良好的環(huán)境適應(yīng)能力、繁殖能力以及生長速率，能在固體培養(yǎng)基上形成單藻落，也可在液體中培養(yǎng)，有“綠色酵母”之稱(謝傳曉等, 2003; 孫曉梅等, 2004; 耿予歡等, 2007)。目前，萊茵衣藻全基因組測序已經(jīng)完成(Merchant, 2007)，已成功建立針對萊茵衣藻線粒體、葉綠體與細(xì)胞核基因組的遺傳操作系統(tǒng)和CRISPR/Cas9介導(dǎo)的基因組精確編輯系統(tǒng)(Greiner, 2017)。此外，目前已建立萊茵衣藻光合作用、呼吸作用、細(xì)胞分裂、養(yǎng)分吸收、輻射、紫外線和化學(xué)誘變下的基因組穩(wěn)定性和適應(yīng)性反應(yīng)、耐重金屬植物修復(fù)、鹽度、水溫、滲透壓和氧化脅迫應(yīng)激等重要生理過程研究的模型(Harris, 2001; Hanikenne, 2003; Hema, 2007; Dimova, 2008)。因此，萊茵衣藻已經(jīng)成為藻類遺傳學(xué)、生物化學(xué)和分子生物學(xué)研究的重要模式物種(徐田枚, 2007; Harper, 1999; Wu, 1997)。

植物程序性細(xì)胞死亡(Programmed cell death)是指與動物細(xì)胞凋亡(Apoptosis)同源的植物細(xì)胞死亡方式，普遍存在于植物發(fā)育和應(yīng)對病原感染、干旱等生物或非生物脅迫過程中(Reape, 2008)。然而，動物細(xì)胞凋亡通路中關(guān)鍵調(diào)控與執(zhí)行基因家族胱冬肽酶(Cysteine aspartic acid specific protease, caspase)家族在植物中并不存在(van Doorn, 2005; Ge, 2016)。研究表明，植物中存在一種caspase同系蛋白metacaspase。Metacaspase是非后生動物完成正常生理學(xué)反應(yīng)所必需的多功能蛋白酶，且植物和原生動物中氧化應(yīng)激誘導(dǎo)的程序性細(xì)胞死亡過程都依賴于metacaspase(Vercammen, 2007)。根據(jù)其結(jié)構(gòu)特征，metacaspase又可以分為type I與typeⅡ兩類(馬聰?shù)? 2012)。其中，type I metacaspase具有富含脯氨酸重復(fù)基序的N末端前結(jié)構(gòu)域，并且包含鋅指結(jié)構(gòu)。而type Ⅱ metacaspase缺乏前結(jié)構(gòu)域，但在大(p20)、小(p10)亞基之間具有較長連接區(qū)。在原生動物和真菌中，目前僅發(fā)現(xiàn)了type I metacaspase，而高等植物基因組中同時具有type I與type Ⅱmetacaspase。Metacaspase活性中心普遍具有保守的組氨酸–半胱氨酸結(jié)構(gòu)，其中半胱氨酸殘基作為水解底物肽鍵的親核體。

近年的研究表明，單細(xì)胞藻類在生物和非生物脅迫條件下，亦可發(fā)生程序性細(xì)胞死亡。萊茵衣藻經(jīng)被用于程序性細(xì)胞死亡研究，并獲得了成功(Moharikar, 2006; Zuo, 2012; Pérezpérez, 2010; Yordanova, 2013; Sirisha, 2014)。全基因組序列預(yù)測顯示，萊茵衣藻基因組中同時存在type I與typeⅡ metacaspase基因，其結(jié)構(gòu)與功能尚不明確。本研究通過巢式PCR克隆獲得萊茵衣藻type I metacaspase ORF序列，利用生物信息學(xué)技術(shù)預(yù)測其氨基酸序列并進(jìn)行同源性分析，繼而利用熒光定量PCR技術(shù)分析type I metacaspase基因在H2O2誘導(dǎo)的萊茵衣藻程序性細(xì)胞死亡過程中的表達(dá)量變化。本研究為更好地闡明植物與微藻程序性細(xì)胞死亡的分子機(jī)制及其生物演化關(guān)系提供理論依據(jù)，同時為高品質(zhì)微藻藻種選育和微藻增養(yǎng)殖工業(yè)及應(yīng)用(何舟等, 2015; 喬洪金等, 2016)提供技術(shù)支持。

1 材料與方法

1.1 藻種培養(yǎng)

野生型萊茵衣藻藻種cc-125 mt+購自美國明尼蘇達(dá)大學(xué)萊茵萊茵衣藻種質(zhì)庫(Chlamycollection, University of Minnesota, 美國)。藻種接種至含100 μmol/L氨芐青霉素的固體TAP培養(yǎng)基平板，挑取單克隆接種于液體TAP培養(yǎng)基中，置于25℃、光照強(qiáng)度2000 lx的光照培養(yǎng)箱振蕩培養(yǎng)(100 r/min)至對數(shù)生長期(OD434 nm=1)，光/暗周期為12 h/12 h。

1.2 萊茵衣藻總RNA提取與cDNA合成

藻種離心收集后，采用液氮反復(fù)凍融法破壁，采用QIAGEN RNeasy Plant Mini Kit試劑盒提取萊茵衣藻總RNA。采用瓊脂糖凝膠電泳法和Qubit 3.0分光光度計鑒定總RNA質(zhì)量與濃度。cDNA合成采用M-MLV反轉(zhuǎn)錄酶試劑盒進(jìn)行。利用萊茵衣藻18 rRNA (GenBank No. AY665726)作為內(nèi)參。

1.3 CrMC1 ORF巢式PCR擴(kuò)增

根據(jù)NCBI GenBank數(shù)據(jù)庫中萊茵衣藻type I metacaspase cDNA序列(GenBank No. XM_001696904)設(shè)計引物，進(jìn)行兩步法巢式PCR克隆，引物序列見表1。使用Nucleospin Gel and PCR Clean-up試劑盒純化第二步PCR產(chǎn)物，并進(jìn)行poly(A)-tailing添加粘末端反應(yīng)，進(jìn)而將其連接到pGEM-T載體中進(jìn)行藍(lán)白斑篩選。篩選后提取質(zhì)粒并送至生工生物工程(上海)有限公司進(jìn)行測序、組裝并輸入BLAST進(jìn)行驗證，證實所獲序列為萊茵衣藻type I metacaspase ORF序列，命名為CrMC1。

表1 萊茵衣藻metacaspase type I ORF克隆引物

Tab.1 Primers for C. reinhardtii metacaspase type I ORF cloning

1.4 生物信息學(xué)分析

利用在線SMS生物軟件(http://www.bio-soft.net/ sms/)，根據(jù)CrMC1 ORF的DNA序列推測CrMC1氨基酸序列，獲得包含405個氨基酸的序列。進(jìn)而利用Vector NTI 11.5軟件ClustalX方法將CrMC1與從NCBI數(shù)據(jù)庫獲得的其他物種metacaspase構(gòu)建系統(tǒng)進(jìn)化樹，物種名與蛋白編號見表2。此外，通過將CrMC1氨基酸序列與自uniprot數(shù)據(jù)庫(http://www.uniprot. org/)獲得的3種擬南芥() type I metacaspase (AtMC1, No. Q7XJE6; AtMC2, No. Q7XJE5; AtMC3, No. Q9FMG1)氨基酸序列的同源比對，預(yù)測其功能域。

1.5 H2O2誘導(dǎo)萊茵衣藻細(xì)胞死亡率測定

將處于生長對數(shù)期(OD434 nm=1)的萊茵衣藻cc125 mt+分別置于實驗組(1 mmol/L H2O2)與對照組進(jìn)行培養(yǎng)，并測定0~480 min內(nèi)的死亡率。死亡率測定采取SYTOX green染料進(jìn)行。利用正置熒光顯微鏡觀察計數(shù)至少200個細(xì)胞，根據(jù)SYTOX green標(biāo)記陽性為死亡細(xì)胞、隱性為存活細(xì)胞進(jìn)行細(xì)胞計數(shù)，并計算死亡率。

表2 進(jìn)化樹所用metacaspase基因序列

Tab.2 Metacaspase gene sequence of phylogenetic tree

1.6 熒光定量PCR

提取不同時間點(diǎn)實驗組與對照組萊茵衣藻總RNA，并反轉(zhuǎn)錄獲得cDNA，利用熒光定量PCR法進(jìn)行CrMC1表達(dá)量分析。熒光定量PCR反應(yīng)使用SensiFAST SYBR No-ROX試劑盒在ABI Prism 7000型熒光定量PCR儀上進(jìn)行。熒光定量PCR所得數(shù)據(jù)利用18S rRNA作為內(nèi)參基因，按照2-DD法處理數(shù)據(jù)。所得數(shù)據(jù)利用Microsoft Excel 2010軟件進(jìn)行平均值、標(biāo)準(zhǔn)差和差異顯著性分析(<0.05)并作圖。CrMC1與內(nèi)參基因18S rRNA (Nguyen, 2011)引物見表3。

表3 熒光定量PCR引物

Tab.3 Primers used in qRT-PCR

2 結(jié)果

2.1 CrMC1基因克隆及進(jìn)化分析

如圖1A所示，本研究通過巢式PCR克隆到萊茵衣藻type I metacaspase基因ORF全長序列，包含987 bp，推測編碼405個氨基酸，命名為CrMC1。為分析CrMC1進(jìn)化關(guān)系，本研究從NCBI數(shù)據(jù)庫中篩選出已分類的植物metacaspase蛋白氨基酸序列，包括擬南芥、小麥()、雞()、挪威云杉()、團(tuán)藻()等(表1)，使用Vector NTI 11.5軟件的ClustalX法進(jìn)行同源性分析，并構(gòu)建系統(tǒng)進(jìn)化樹(NJ)。如圖1B所示，系統(tǒng)進(jìn)化樹中的19個metacaspase成員形成了2個大分支，其中，CrMC1與所有已知type I metacaspase位于同一分支內(nèi)，故可推測CrMC1屬于type I metacaspase。此外，在type I metacaspase分支中，CrMC1與團(tuán)藻目的metacaspase具有最高相似性，與植物例如擬南芥type I metacaspase AtMC2親緣關(guān)系較遠(yuǎn)，提示其位于生物演化的初級地位。

2.2 CrMC1蛋白結(jié)構(gòu)域預(yù)測

蛋白多序列比對分析顯示(圖2)，CrMC1具有metacaspase家族典型的、高度保守的p20 (氨基酸44~ 195)結(jié)構(gòu)域、p10 (氨基酸229~328)結(jié)構(gòu)域以及中間連接區(qū)(氨基酸196~228)。與擬南芥type I metacaspase 蛋白AtMC1/2/3比較，雖然CrMC1的N末端區(qū)域較為簡單，然而其脯氨酸含量較高，為典型的富含脯氨酸區(qū)域(23.2%)，符合type I metacaspase的結(jié)構(gòu)特征。CrMC1蛋白酶活性中心包含高度保守的組氨酸(132) (圖2*處)與半胱氨酸(188) (圖2#處)結(jié)構(gòu)，推測其具有已知metacaspase類似的蛋白酶活性。

圖1 CrMC1結(jié)構(gòu)分析

A.通過利用SMS生物軟件(http://www.bio-soft.net/sms/)推測CrMC1氨基酸序列；B. 利用Vector NTI 11.5軟件ClustalX方法將CrMC1與從NCBI數(shù)據(jù)庫獲得的其他物種metacaspase構(gòu)建系統(tǒng)進(jìn)化樹，物種名與蛋白編號見表2

A.CrMC1 ORF was deduced to CrMC1 protein sequence using online SMS software (http://www.bio-soft.net/sms/); B. Phylogenetic tree was constructed using metacaspase protein sequence of different species obtained from NCBI database. Latin names and protein IDs are presented in Table 2

圖2 CrMC1蛋白質(zhì)結(jié)構(gòu)預(yù)測

利用Vector NTI 11.5軟件ClustalX方法將CrMC1與自uniprot數(shù)據(jù)庫(http://www.uniprot.org/)獲得的3種擬南芥I型metacaspase (AtMC1, NO. Q7XJE6; AtMC2, NO. Q7XJE5; AtMC3, NO. Q9FMG1)氨基酸序列進(jìn)行同源比對。圖中藍(lán)色背景標(biāo)示為p20結(jié)構(gòu)域，紅色背景標(biāo)示為p10結(jié)構(gòu)域，黑框標(biāo)示區(qū)域為連接區(qū)。活性中心分別以*和#標(biāo)出

CrMC1 was aligned with type I metacaspase from Arabidopsis: AtMC1, NO. Q7XJE6; AtMC2, NO. Q7XJE5; AtMC3, NO. Q9FMG1. Arabidopsis sequences were obtained from uniprot database. The domain of p20, p10 and internal linker is highlighted in blue background, red background and black frame, respectively. The conserved His-Cys residues are labelled by * and # respectively

2.3 萊茵衣藻CrMC1參與H2O2誘導(dǎo)細(xì)胞死亡調(diào)控

由圖3A可見，1 mmol/L H2O2處理可誘導(dǎo)處于對數(shù)生長期的萊茵衣藻出現(xiàn)明顯的細(xì)胞死亡現(xiàn)象。根據(jù)Vavilala等(2015)報道，H2O2誘導(dǎo)的萊茵衣藻細(xì)胞死亡方式為程序性細(xì)胞死亡。本研究進(jìn)而利用熒光定量PCR方法檢測處于對數(shù)生長期的萊茵衣藻CrMC1基因在1 mmol/L H2O2誘導(dǎo)后1~8 h內(nèi)表達(dá)水平的變化情況。如圖3B所示，1 mmol/L H2O2誘導(dǎo)后，萊茵衣藻CrMC1基因的表達(dá)量自1 h起顯著高于對照組(<0.05)，2 h后達(dá)到最高，約為對照組1.77倍，5 h時下降至對照組同一水平，與程序性細(xì)胞死亡發(fā)生時間基本同步，推測CrMC1處于萊茵衣藻程序性細(xì)胞死亡調(diào)控通路的上游。

圖3 CrMC1參與H2O2誘導(dǎo)的萊茵衣藻程序性細(xì)胞死亡調(diào)控

A. H2O2處理后1~480 min內(nèi)萊茵衣藻細(xì)胞死亡率。萊茵衣藻培養(yǎng)與H2O2處理如前所述；B. CrMC1基因在H2O2誘導(dǎo)的萊茵衣藻程序性細(xì)胞死亡中的表達(dá)量變化。實驗均進(jìn)行3次取平均值

A. The mortality ofcells within 1~480 min after H2O2treatment.culture and H2O2treatment as previously described; B. CrMC1 gene expression changes in H2O2-induced PCD in. Each experiment was performed three times and averaged

3 討論

Madeo等(2002)首次報道了使用酵母Yca1突變菌株在氧化應(yīng)激導(dǎo)致的細(xì)胞死亡對metacaspase的依賴性，證明Yca1在酵母細(xì)胞中參與病毒毒素和非生物相關(guān)因素應(yīng)激以及細(xì)胞衰老過程中的細(xì)胞死亡(Madeo, 2009)。此外，還有研究表明，脂毒性應(yīng)激(Low, 2008)和肌醇饑餓(Guérin, 2009)等部分酵母細(xì)胞致死條件下，metacaspase是酵母實現(xiàn)細(xì)胞死亡的必要的條件；白念珠菌的metacaspase，CaMCA1，參與介導(dǎo)氧化應(yīng)激誘導(dǎo)的細(xì)胞死亡(Cao, 2009)；而構(gòu)巢曲霉的2個metacaspase的其中之一，CasA在ER應(yīng)激相關(guān)的細(xì)胞死亡起促進(jìn)作用(Colabardini, 2010)。

目前，在模式生物擬南芥中分別鑒定出3個type I metacaspase基因(AtMC1~3)與6個typeⅡ metacaspase基因(AtMC4~9)(Vercammen, 2004)。Type I metacaspase AtMC1顯示為擬南芥中HR細(xì)胞死亡的正調(diào)節(jié)物，而AtMC2則能拮抗AtMC1的這種細(xì)胞死亡效應(yīng)。而且AtMC2作為抗凋亡調(diào)節(jié)劑，其蛋白水解活性是顯性的。通過對2種metacaspase的遺傳操作，幾乎可以使植物細(xì)胞內(nèi)的免疫受體消除HR活性(Coll, 2010)。與高等植物相似，萊茵衣藻中同時存在1個type I metacaspase基因與1個typeⅡ metacaspase基因。由此推測，與酵母相比，萊茵衣藻程序性細(xì)胞死亡調(diào)控與高等植物具有更高的同源性。因此，對萊茵衣藻metacaspase基因及其功能的研究，對更好地闡釋植物程序性細(xì)胞死亡調(diào)控通路以及type I與typeⅡ metacaspase基因相互作用有著極為重要的意義。

本研究首次報道了萊茵衣藻type I metacaspase基因ORF序列及其參與調(diào)控H2O2誘導(dǎo)程序性細(xì)胞死亡作用。通過巢式PCR從野生型萊茵衣藻藻種中克隆到1個type I metacaspase基因并命名為CrMC1。序列分析表明，CrMC1具有type I metacaspase特征性的N末端脯氨酸富含區(qū)、保守的p20與p10結(jié)構(gòu)域以及組氨酸–半胱氨酸活性中心結(jié)構(gòu)，推測其在演化上與已知高等植物metacaspase具同源性。Type I metacaspase在高等植物與酵母程序性細(xì)胞死亡中所扮演的角色尚存在爭議。例如，AtMC2表達(dá)量在擬南芥程序性細(xì)胞死亡過程中調(diào)高而AtMC1表達(dá)量調(diào)低或無變化。本研究的實驗數(shù)據(jù)證實，與AtMC2類似，CrMC1表達(dá)量在過氧化物脅迫導(dǎo)致的萊茵衣藻程序性細(xì)胞死亡早期顯著提高，證實其參與早期程序性細(xì)胞死亡的調(diào)控。

綜上所述，本研究結(jié)果初步描述了萊茵衣藻中type I metacaspase基因的結(jié)構(gòu)與功能，為植物程序性細(xì)胞死亡研究和藻類增養(yǎng)殖工業(yè)提供了理論基礎(chǔ)和技術(shù)支持。

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Cloning of the Metacase Type I Gene ofand Its Involvement in the Regulation of Programmed Cell Death

ZHANG Hengyu1, LIU Yiqun2, GE Yuan1①

(1. College of Marine Life Science, Ocean University of China, Qingdao 266003; 2. College of Fisheries, Ocean University of China, Qingdao 266003)

The metacaspase is a cysteine protease with the substrate of arginine/lysine, specifically found in protozoa, fungi, and plants. The metacaspase is generally believed to be closely related to the caspase of the metazoan. Previous studies have shown that metacaspases can be divided into type I and typeⅡ according to the differences in their structural characteristics, and both types have been found to be involved in the regulation and the control of programmed cell death in various plants and protozoa. Based on the information of the sequence of thetype I metacaspase cDNA, obtained from the Genbank database (Genbank NO. XM_001696904), this study was able to use the method of two-step nested PCR cloning to retrive the Open Reading Frame (ORF) of the sequence of thetype I metacaspase cDNA. The ORF sequence, mentioned above, was named as the CrMC1 in this study. The length of the CrMC1 ORF, in its entirety, is 987 bp, from which can be inferred that there are 405 amino acids encoded in this sequence. By the method of homologous sequence comparative analysis with the already known type I metacaspase protein, it was then found that the CrMC1 has conservative p20, p10, linker domain, and arginine and cysteine active center sites. Furthermore, this study is also able to show that, during the H2O2induced programmed cell death of, the gene transcripts of the CrMC1 was increased at a statistically significant level (<0.05). The gene transcripts of the CrMC1 then reached its peak after 2 h. Eventually, the gene transcripts of the CrMC1 decreased to the same level as it was in the control group at 3 h. In conclusion, the results as discussed above have indicated the following: theCrMC1 was involved in the regulation and the control of the H2O2induced programmed cell death of.

Metacaspase;; Programmed cell death

S9

A

2095-9869(2019)06-0131-07

10.19663/j.issn2095-9869.20180507002

http://www.yykxjz.cn/

張恒宇, 柳逸群, 葛源. 萊茵衣藻type I metacaspase基因克隆及其參與調(diào)控程序性細(xì)胞死亡研究. 漁業(yè)科學(xué)進(jìn)展, 2019, 40(6): 131–137

Zhang HY, Liu YQ, Ge Y. Cloning of the metacase type I gene ofand its involvement in the regulation of programmed cell death. Progress in Fishery Sciences, 2019, 40(6): 131–137

* 863計劃生物醫(yī)藥領(lǐng)域青年科學(xué)家專項基金(2015AA020903)和青島市科技計劃基礎(chǔ)研究項目(13-1-4-253-jch)共同資助 [This work was supported by China National High-Tech Research and Development Programme (863 Programme) (2015AA020903), and Qingdao Municipal Science and Technology Project Basic Science Research Programme (13-1-4-253-jch)]. 張恒宇, E-mail: 854636605@qq.com

葛 源，E-mail: geyuan@ouc.edu.cn

2018-05-07,

2018-05-17

GE Yuan, E-mail: geyuan@ouc.edu.cn

(編輯 馮小花)