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大花杓蘭親環(huán)素基因的克隆及生物信息學(xué)分

2019-09-10 07:22付亞娟張劍劉歡侯曉強(qiáng)
廣西植物 2019年5期
關(guān)鍵詞:序列分析基因克隆

付亞娟 張劍 劉歡 侯曉強(qiáng)

摘 要:親環(huán)素是一個(gè)多基因家族,在植物生命活動(dòng)中發(fā)揮著重要的作用。該研究以大花杓蘭(Cypripedium macranthum)為材料,采用RT-PCR技術(shù)克隆到1個(gè)親環(huán)素基因(CyP),并對(duì)其進(jìn)行生物信息學(xué)分析。結(jié)果表明:大花杓蘭CyP基因的開放閱讀框序列為525 bp,命名為CmCyP(GenBank登錄號(hào)為MH411125),編碼174個(gè)氨基酸。預(yù)測(cè)CmCyP蛋白是一個(gè)位于細(xì)胞質(zhì)、相對(duì)分子量約為18 kD、理論pI為8.73、無信號(hào)肽、跨膜結(jié)構(gòu)域的親水性蛋白質(zhì)。磷酸化和糖基化位點(diǎn)預(yù)測(cè)分析發(fā)現(xiàn),CmCyP蛋白存在18個(gè)潛在的磷酸化位點(diǎn)和2個(gè)潛在的糖基化位點(diǎn)。蛋白保守結(jié)構(gòu)域預(yù)測(cè)分析發(fā)現(xiàn),CmCyP蛋白包含一個(gè)高度保守的肽脯氨酰順反異構(gòu)酶結(jié)構(gòu)域,屬于單結(jié)構(gòu)域親環(huán)素。對(duì)二級(jí)結(jié)構(gòu)進(jìn)行預(yù)測(cè)分析發(fā)現(xiàn),CmCyP蛋白中存在無規(guī)卷曲70個(gè)、延伸鏈56個(gè)、α-螺旋23個(gè)、β-折疊25個(gè),這4種結(jié)構(gòu)元件在三級(jí)結(jié)構(gòu)中也有體現(xiàn)。系統(tǒng)進(jìn)化樹結(jié)果顯示,大花杓蘭CmCyP蛋白與鐵皮石斛(Dendrobium catenatum)和萬帶蘭(Vanda hybrid cultivar)的CyP蛋白的親緣關(guān)系較近。該研究首次克隆了大花杓蘭親環(huán)素基因(CmCyP),為進(jìn)一步探討CmCyP基因的生物學(xué)功能奠定了基礎(chǔ)。

關(guān)鍵詞: 大花杓蘭, RT-PCR, 基因克隆, 系統(tǒng)發(fā)育樹, 序列分析

中圖分類號(hào):Q949.95

文獻(xiàn)標(biāo)識(shí)碼:A

文章編號(hào):1000-3142(2019)05-0633-08

Cloning and bioinformatics analysis of cyclophilin gene(CmCyP) from Cypripedium macranthum

FU Yajuan1,2, ZHANG Jian3, LIU Huan1, HOU Xiaoqiang1,2*

( 1. College of Life Sciences, Langfang Normal University, Langfang 065000, Hebei, China; 2. Edible and MedicinalFungi Research and Development Center of Hebei Universities, Langfang 065000, Hebei, China;3. Langfang Polytechnic Institute,Langfang 065000, Hebei, China )

Abstract:Cyclophilin is a multi-gene family that plays an important role in plant biology. A cyclophilin gene in Cypripedium macranthumnamed as CmCyP (GenBank Accession No. MH411125), was successfully cloned by RT-PCR, and then bioinformatics analysis were carried out. The results were as follows: CmCyP protein was predicted as a stable hydrophilic protein composed of 174 amino acids, without signal peptide and transmembrane domain. Its relative molecular mass was approximately 18 kD, and a predicated isoelectric point (pI) was 8.73.

Phosphorylation and glycosylation site prediction analysis revealed that eighteenpotential phosphorylation sites and two potential glycosylation sites were found in CmCyP protein. Protein domain prediction revealed that CmCyP contained a highly conserved peptide prolyl cis-trans isomerase domain, belonging to single domain of cyclophilin. The secondary structure of CmCyP was abundant in random coils (70) and extension chains (56), while was less in α-helices (23) and β-turns (25). Phylogenetic tree analysis indicates that the genetic relationship of CmCyP in C. macranthumwith CyPs from Dendrobium catenatumand Vanda hybrid cultivar is relatively close. In conclusion, the CmCyPgene was firstly cloned fromCypripedium macranthum, which provides for further study on biology function of CmCyPgene.

Key words: Cypripedium macranthum, RT-PCR, gene cloning, phylogenetic tree, sequence analysis

親環(huán)素(Cyclophilin, CyP)是一類普遍存在于原核和真核生物體內(nèi)、在進(jìn)化上非常保守的多功能蛋白質(zhì)(Galat,1999;王保明和譚曉風(fēng),2008)。Gasser et al.(1990)首次從番茄(Lycopersicon esculentum)、玉米(Zea mays)和油菜(Brassica napus)中分離到親環(huán)素蛋白cDNA序列,并在大腸桿菌中成功表達(dá)。隨后,親環(huán)素基因相繼在擬南芥(Arabidopsis thaliana)(Chou & Gasser,1997)、水稻(Oryza sativa)(Lee et al.,2015)、小麥(Triticum aestivum)(Kaur et al.,2016)、棉花(Gossypium hirsutum)(吳莉等,2012)等多種植物中被鑒定和分離。親環(huán)素除一般生物學(xué)功能,如作為分子伴侶發(fā)揮肽脯氨酰順反異構(gòu)酶活性、參與DNA降解的調(diào)控及植物葉綠體的氧化還原調(diào)節(jié)等(童惠姍等,2017),還在植物激素信號(hào)轉(zhuǎn)導(dǎo)(Cheong et al.,2017;Liebthal et al.,2016;Ma et al.,2013)、逆境脅迫應(yīng)答(Ahn et al.,2010;Kumari et al.,2009,2013;趙麗等,2018)、抗病原菌的防御反應(yīng)(Kumari et al.,2001;Ye & Ng,2002)等過程中發(fā)揮重要作用。

大花杓蘭(Cypripedium macranthum)為蘭科杓蘭屬多年生草本植物。除了較高的觀賞價(jià)值,大花杓蘭還具有一定的藥用價(jià)值,可用于全身浮腫、風(fēng)濕腰腿痛、跌打損傷等疾病的治療(陳心啟和吉占和,2003)。基于本課題組前期大花杓蘭轉(zhuǎn)錄組數(shù)據(jù)分析,發(fā)現(xiàn)了一條與植物親環(huán)素基因有較高同源性的序列,但有關(guān)大花杓蘭親環(huán)素的相關(guān)研究目前尚未見有報(bào)道。因此,本研究采用RT-PCR首次克隆了大花杓蘭親環(huán)素基因(CmCyP),并對(duì)其編碼的蛋白質(zhì)序列進(jìn)行生物信息學(xué)分析,旨在為CmCyP基因的生物學(xué)功能的研究奠定理論基礎(chǔ)。

1 材料與方法

1.1 材料

1.1.1 材料 本研究所用的材料為大花杓蘭(Cypripedium macranthum)的根;大腸桿菌(Escherichia coli)TOP10為本實(shí)驗(yàn)室保存。

1.1.2 試劑及試劑盒 植物RNA提取試劑盒(Omega,USA)購自上海索寶生物科技有限公司;BeyoRTTMcDNA第一鏈合成試劑盒購自上海碧云天生物技術(shù)有限公司;即用型PCR擴(kuò)增試劑盒、SanPrep柱式PCR產(chǎn)物純化試劑盒、LB Broth Agar和LB Broth購自生工生物工程(上海)股份有限公司(簡(jiǎn)稱上海生工);pGM-T克隆試劑盒、DNA Marker購自天根生化科技(北京)有限公司;IPTG、X-Gal購自北京華越洋生物科技有限公司。

1.2 方法

1.2.1 大花杓蘭RNA的提取及cDNA第一鏈的合成 采用植物RNA提取試劑盒(Omega,USA)提取大花杓蘭根總RNA,其操作按照試劑盒說明書進(jìn)行,RNA的完整性通過1%瓊脂糖凝膠電泳進(jìn)行檢測(cè)。利用BeyoRTTMcDNA第一鏈合成試劑盒(碧云天,上海)合成cDNA。RT-PCR反應(yīng)體系:Recation Buffer (5X) 4 μL,RNA模板1 μL,引物Oligo(dT)18 (0.5 μg·μL<SUP>-1</SUP>)1 μL,RNase Inhibitor (20 U·μL<SUP>-1</SUP>) 1 μL,dNTP Mix (10 mmol·L<SUP>-1</SUP>) 1 μL,M-MLV反轉(zhuǎn)錄酶(200 U·μL<SUP>-1</SUP>)1 μL;加ddH2O至20 μL。RT-PCR反應(yīng)程序?yàn)?2 ℃,60 min;80 ℃,10 min。將cDNA第一鏈置于-20 ℃冰箱中保存?zhèn)溆谩?/p>

1.2.2 大花杓蘭CmCyP基因的克隆和測(cè)序 基于課題組前期獲得的大花杓蘭轉(zhuǎn)錄組數(shù)據(jù),采用Oligo 6軟件設(shè)計(jì)一對(duì)特異性引物用于CmCyP基因的PCR擴(kuò)增,正向引物CmCyPF(5′-CCGGAATTCATGGCAGCGACCA-3′,加粗的核苷酸序列為保護(hù)堿基,下劃線的核苷酸為EcoRⅠ酶切位點(diǎn))和反向引物CmCyPR(5′-CACCGCTCGAGCTAAGAGAGCTG-3′,加粗的核苷酸序列為保護(hù)堿基,下劃線的核苷酸為XhoⅠ酶切位點(diǎn))。PCR反應(yīng)體系:2×PCR Master mix 12.5 μL,cDNA 2 μL,正向和反向引物(10 μmol·L<SUP>-1</SUP>)各1 μL;加ddH2O至25 μL。PCR程序:94 ℃預(yù)變性3 min;94 ℃變性30 s,57 ℃復(fù)性30 s,72 ℃延伸40 s,共30個(gè)循環(huán);72 ℃最后延伸7 min。PCR產(chǎn)物經(jīng)1.0%瓊脂糖凝膠電泳檢測(cè)后,利用PCR產(chǎn)物純化試劑盒對(duì)其進(jìn)行純化。純化的PCR產(chǎn)物與克隆載體pGM-T進(jìn)行連接,并轉(zhuǎn)化E. coliTOP10感受態(tài)細(xì)胞。通過藍(lán)白斑篩選和菌落PCR鑒定出的陽性重組子送至上海生工進(jìn)行測(cè)序,測(cè)序引物為載體通用引物T7。

1.2.3 大花杓蘭CmCyP蛋白系統(tǒng)發(fā)育分析 測(cè)序結(jié)果用Chromas軟件打開,觀察峰圖,排除雙峰序列,獲得良好DNA序列;采用DNAstar去除載體冗余序列;應(yīng)用NCBI的Blastn與GenBank中已有序列進(jìn)行同源性分析,初步確定分離的序列即為大花杓蘭CyP基因?;谠诰€基因預(yù)測(cè)軟件ORF Finder對(duì)CmCyP基因編碼的氨基酸序列進(jìn)行推測(cè),并通過NCBI的Blastp程序在氨基酸水平上對(duì)大花杓蘭CmCyP蛋白序列進(jìn)行同源性分析。用Clustal X2軟件對(duì)大花杓蘭CmCyP蛋白及與其相似性較高的其它植物親環(huán)蛋白進(jìn)行多序列比對(duì)分析,并使用Mega 5.1中的鄰接法來構(gòu)建系統(tǒng)發(fā)育樹。

1.2.4 大花杓蘭CmCyP的生物信息學(xué)分析 用在線軟件ProtParam(http://web.expasy.org/protparam/)對(duì)CmCyP理化性質(zhì)進(jìn)行預(yù)測(cè);SignaIP4.1 Server(http://www.cbs.dtu. dk/services/SignalP/)分析CmCyP信號(hào)肽;Psort(http://www.genscript.com/psort.html)對(duì)CmCyP蛋白進(jìn)行亞細(xì)胞定位;TMpred(https://embnet.vital-it.ch/software/TMPRED_form.html)預(yù)測(cè)跨膜結(jié)構(gòu)域;Protscale(http://web.expasy.org/protscale/)對(duì)其進(jìn)行親水性和疏水性分析;用NetPhos 3.1(http://www.cbs.dtu.dk/services/NetPhos/)預(yù)測(cè)CmCyP蛋白潛在的磷酸化位點(diǎn);用NetOGlyc 4.0(http://www.cbs.dtu.dk/services/NetOGlyc/)進(jìn)行CmCyP蛋白糖基化位點(diǎn)預(yù)測(cè);通過InterProScan(http://www.ebi.ac.uk/interpro/search/sequence-search)預(yù)測(cè)大花杓蘭CmCyP蛋白的保守結(jié)構(gòu)域及功能位點(diǎn)。用在線軟件SOPMA(https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.htm)對(duì)大花杓蘭CmCyP的二級(jí)結(jié)構(gòu)進(jìn)行預(yù)測(cè);用在線軟件SWISS-MODEL(http://swissmodel.expasy.org/),基于同源建模法對(duì)該蛋白的三級(jí)結(jié)構(gòu)進(jìn)行預(yù)測(cè)。

2 結(jié)果與分析

2.1 CmCyP基因的RT-PCR擴(kuò)增

以大花杓蘭根總RNA反轉(zhuǎn)錄獲得的cDNA為模板,CmCyPF和CmCyPR為特異性引物,進(jìn)行PCR擴(kuò)增。PCR產(chǎn)物經(jīng)1.0%瓊脂糖凝膠電泳檢測(cè)(圖1)。從圖1可以看出,PCR擴(kuò)增得到1條清晰明亮、約500 bp的條帶,其大小與CmCyP目的基因理論值基本相符。

2.2 CmCyP基因的克隆及測(cè)序

將純化的PCR產(chǎn)物與載體pGM-T進(jìn)行連接、轉(zhuǎn)化E. coliTOP10。對(duì)轉(zhuǎn)化平板上長出的白色單菌落進(jìn)行菌落PCR檢測(cè),電泳結(jié)果如圖2所示。菌落PCR擴(kuò)增得到1條大小約500 bp的條帶,與預(yù)期大小一致。將菌落PCR鑒定的陽性重組子送至上海生工進(jìn)行測(cè)序,獲得大花杓蘭CmCyP基因的ORF序列,大小為525 bp(提交至GenBank,登錄號(hào)為MH411125),編碼174個(gè)氨基酸(圖3)。

2.3 CmCyP蛋白的系統(tǒng)發(fā)育分析

將大花杓蘭CmCyP基因編碼的氨基酸序列在GenBank進(jìn)行Blastp搜索,發(fā)現(xiàn)大花杓蘭CmCyP與多種植物親環(huán)蛋白具有較高的序列相似性。選取與大花杓蘭CmCyP同源性較高的8條蛋白序列,鐵皮石斛(Dendrobium catenatumXP_020689035)、芝麻(Sesamum indicumXP_011074689)、陸地棉(Gossypium hirsutum ACT63839)、西洋長春藤(Hedera helixAPV45518)、三七(Panax notoginsengAIX10768)、大麻槿(Hibiscus cannabinusAQA28693)、落花生(Arachis diogoi ABY57946)和萬帶蘭(Vanda hybrid cultivar ADG34846),構(gòu)建系統(tǒng)發(fā)育樹(圖4)。圖4結(jié)果表明,來自9個(gè)不同植物的CmCyP蛋白聚成2個(gè)分支。大花杓蘭親環(huán)蛋白CmCyP與鐵皮石斛(Dendrobium catenatum)和萬帶蘭(Vanda hybrid cultivar)CyP處于同一分支上,親緣關(guān)系較近。這與他們均隸屬蘭科植物的進(jìn)化及分類地位較一致。

2.4 CmCyP蛋白的生物信息學(xué)分析

采用一系列在線軟件對(duì)大花杓蘭CmCyP基因編碼蛋白進(jìn)行生物信息學(xué)預(yù)測(cè)分析。推測(cè)CmCyP蛋白的分子式為C803H1261N227O246S9,相對(duì)分子量為18.32 kD,理論等電點(diǎn)(pI)為8.73。預(yù)測(cè)大花杓蘭CmCyP蛋白位于細(xì)胞質(zhì)、不含信號(hào)肽,無跨膜結(jié)構(gòu)域、穩(wěn)定的親水性蛋白。

采用InterProScan對(duì)CmCyP蛋白的保守結(jié)構(gòu)域進(jìn)行預(yù)測(cè)分析(圖5)。圖5結(jié)果表明,大花杓蘭CmCyP蛋白含4種保守結(jié)構(gòu)域,分別在氨基酸序列的3~173位是親環(huán)素樣蛋白超家族(IPR029000);6~174位是親環(huán)素型肽脯氨酰順反異構(gòu)酶(IPR024936);26~41、62~74、105~132和133~148為親環(huán)素型肽脯氨酰順反異構(gòu)酶的結(jié)構(gòu)域(IPR002130);57~74位是親環(huán)素型肽脯氨酰順反異構(gòu)酶的保守位點(diǎn)(IPR0020892)。CmCyP蛋白的活性位點(diǎn)為63H、64R、69F、120Q、122F和130W(圖3)。應(yīng)用NetPhos 3.1程序?qū)mCyP蛋白的磷酸化位點(diǎn)進(jìn)行預(yù)測(cè),結(jié)果表明該蛋白序列存在18個(gè)潛在的磷酸化位點(diǎn),其中9個(gè)絲氨酸(Ser)位點(diǎn)、8個(gè)蘇氨酸(Thr)位點(diǎn)和1個(gè)酪氨酸(Tyr)位點(diǎn)。這些磷酸化位點(diǎn)主要能夠被p38MAPK、unsp、SRC、GSK3、PKA、PKC、PKG、CKⅡ等蛋白激酶磷酸化。用NetOGlyc 4.0 進(jìn)行糖基化位點(diǎn)預(yù)測(cè)分析,發(fā)現(xiàn)CmCyP蛋白含2個(gè)潛在的糖基化位點(diǎn)。

利用SOPMA預(yù)測(cè)大花杓蘭CmCyP蛋白二級(jí)結(jié)構(gòu),結(jié)果如圖6所示。該蛋白含有13.37%的α-螺旋(Alpha helix),31.98%的延伸鏈(Extended strand),14.53%的β-轉(zhuǎn)角(Beta turn)和40.12%的無規(guī)則卷曲(Random coil)。采用SWISS-MODEL在線工具,以甜橙(Citrus sinensis)(PDB ID:4jjm.1)為模板,序列相似性為57%,在5~174位氨基酸同源建模,預(yù)測(cè)大花杓蘭CmCyP的三級(jí)結(jié)構(gòu)(圖7)。從預(yù)測(cè)的三維模型可以看出,大花杓蘭CmCyP蛋白主要由無規(guī)則卷曲、延伸鏈、α-螺旋和β-轉(zhuǎn)角組成,與二級(jí)結(jié)構(gòu)預(yù)測(cè)結(jié)果基本一致。

3 討論與結(jié)論

親環(huán)素是一個(gè)多基因家族,其中擬南芥有31個(gè)CyP基因、水稻29個(gè)CyP基因、大豆62個(gè)CyP基因(童惠姍等,2017)。CyP基因目前在很多植物中已被鑒定,但其功能只在擬南芥和水稻中有較深入研究。Kim et al.(2012)研究發(fā)現(xiàn)水稻親環(huán)素基因(OsCYP20-2)能增強(qiáng)擬南芥和煙草的抗旱性。水稻OsCYP19-4基因過表達(dá)能提高水稻生物量和耐冷能力(Hwa et al.,2016;Lee et al.,2016)。在擬南芥中,過表達(dá)木豆親環(huán)素基因(CcCyP)的植株對(duì)冷、熱、鹽、干旱等逆境脅迫均具有較好的耐受性(Sekhar et al.,2010)。Pogorelko et al. (2014)研究證實(shí)擬南芥親環(huán)素AtCYP19參與其對(duì)抗病原細(xì)菌丁香假單胞菌(Pseudomonas syringae)的防御反應(yīng)。水稻親環(huán)素蛋白CyP1通過影響稻瘟病菌(Magnaporthe grisea)附著孢形成和菌絲生長發(fā)揮其抗菌作用(Viaud et al.,2002)。Lee et al.(2007)研究發(fā)現(xiàn)大白菜親環(huán)素對(duì)多種病原真菌(如Botrytis cinerea,Trichoderma harzianum,T. viride,Rhizoctonia solani,F(xiàn)usarium solani, F. oxysporum)均有不同程度的抑菌作用。Zhang et al.(2017)證實(shí)人參親環(huán)素pgCyP對(duì)植物病原真菌Phytophthora cactorum具有較強(qiáng)的抑制作用,但對(duì)Rhizoctonia solani,F(xiàn)usarium solani和Botrytis cinerea沒有抑菌作用。

大花杓蘭為兼具觀賞和藥用價(jià)值的蘭科植物,其基因組中含多少個(gè)親環(huán)素基因,他們是否存在可變剪接?生物學(xué)功能如何?這些基礎(chǔ)科學(xué)問題的研究目前國內(nèi)外尚未見報(bào)道。因此,基于前期大花杓蘭轉(zhuǎn)錄組數(shù)據(jù),本研究采用RT-PCR首次克隆了大花杓蘭親環(huán)素基因,命名為CmCyP。測(cè)序結(jié)果表明,大花杓蘭CmCyP基因cDNA全長525 bp,編碼174個(gè)氨基酸。蛋白多序列比對(duì)分析表明,CmCyP在氨基酸水平上與多種植物的CyP具有較高的同源性。系統(tǒng)進(jìn)化樹結(jié)果顯示,大花杓蘭CmCyP與鐵皮石斛(Dendrobium catenatum)和萬帶蘭(Vanda hybrid cultivar)的CyP蛋白的親緣關(guān)系較近,這與他們同屬于蘭科植物的分類地位相一致;生物信息在線分析,預(yù)測(cè)CmCyP相對(duì)分子量為18.32 kD,理論等電點(diǎn)為8.73,該蛋白為存在于細(xì)胞質(zhì)、無信號(hào)肽和跨膜結(jié)構(gòu)域、穩(wěn)定的親水性蛋白。蛋白保守結(jié)構(gòu)域預(yù)測(cè)表明,大花杓蘭CmCyP屬于親環(huán)素樣蛋白超家族,含有一個(gè)高度保守的肽脯氨酰順反異構(gòu)酶結(jié)構(gòu)域,屬于單結(jié)構(gòu)域親環(huán)素。CmCyP蛋白包含18個(gè)潛在的磷酸化位點(diǎn)和2個(gè)潛在的糖基化位點(diǎn)。鑒于CmCyP蛋白磷酸化位點(diǎn)較多,推測(cè)其在大花杓蘭中生物學(xué)功能的發(fā)揮可能與激酶磷酸化有關(guān)。采用SOPMA預(yù)測(cè)CmCyP蛋白二級(jí)結(jié)構(gòu),發(fā)現(xiàn)該蛋白主要由延伸鏈和無規(guī)則卷曲組成,α-螺旋和β-轉(zhuǎn)角相對(duì)較少。本研究工作的開展,對(duì)下一步探究大花杓蘭親環(huán)素基因的生物學(xué)功能具有重要意義。大花杓蘭CmCyP也是逆境脅迫相關(guān)蛋白嗎?CmCyP的表達(dá)和積累與菌根真菌侵染大花杓蘭根系過程相關(guān)嗎?CmCyP蛋白參與對(duì)抗植物病原菌的防御反應(yīng)嗎?這些問題有待于進(jìn)一步研究探討。

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