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1、青蝦冷休克蛋白Y-box編碼基因的cDNA全長(zhǎng)克隆與表達(dá)分析孫盛明傅洪拓戈賢平朱健喬慧金舒博張文宜中國水產(chǎn)科學(xué)研究院淡水漁業(yè)研究中心農(nóng)業(yè)部淡水漁業(yè)與種質(zhì)資源利用重點(diǎn)實(shí)驗(yàn)室南京農(nóng)業(yè)大學(xué)無錫漁業(yè)學(xué)院摘要:為研究冷休克蛋白Y-box基因在青蝦應(yīng)答環(huán)境脅迫過程中所起的調(diào)控作用,實(shí)驗(yàn)應(yīng)用RACEPCR技術(shù)首次克隆了青蝦的冷休克蛋白Y-box基因全長(zhǎng)cDNA序列,并利用在線軟件對(duì)其序列特征進(jìn)行生物信息學(xué)分析;采用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)其在青蝦不同組織及環(huán)境脅迫過程中的表達(dá)變化特征進(jìn)行分析。青蝦冷休克蛋白Y-box基因cDNA全長(zhǎng)
2、1501bp,包括84bp的5’末端非翻譯區(qū)(UTR),876bp的開放閱讀框(ORF),541bp的3’UTR,開放閱讀框編碼291個(gè)氨基酸。氨基酸相似度比對(duì)顯示,青蝦冷休克蛋白Y-box基因富含高度保守的冷休克結(jié)構(gòu)域。系統(tǒng)進(jìn)化樹分析顯示,青蝦冷休克蛋白Y-box基因與水蚤等節(jié)肢動(dòng)物冷休克Y-box聚類一支,具有最近的親緣關(guān)系。熒光定量PCR檢測(cè)顯示,冷休克蛋白Y-box基因在青蝦不同組織中均有表達(dá),其表達(dá)量在肝胰腺組織中最高,使用熒光定量PCR檢測(cè)青蝦冷休克蛋白Y-box基因在低溫脅迫和恢復(fù)條件下在肝胰腺屮的mR
3、A時(shí)空表達(dá)情況,結(jié)果顯示,與對(duì)照組相比冷休克蛋Y-box在肝胰腺中的表達(dá)量分別在低溫和低氧脅迫3,6和12h出現(xiàn)了顯著上調(diào),而在恢復(fù)刺激后其表達(dá)量與對(duì)照組差異不顯著。此外,本實(shí)騎對(duì)丫-box進(jìn)行了原核表達(dá),為進(jìn)一步研究Y-box基因的功能奠定了基礎(chǔ)。關(guān)鍵詞:青蝦;冷休克蛋口;低溫;低氧;原核表達(dá);作者簡(jiǎn)介:傅洪拓,E-ina訂:fuht@ffrc.cn收稿日期:2016-08-31基金:國家自然科學(xué)基金(31402280,31672633)Molecularcloningandexpressionanalysisofc
4、oldshockproteinY~boxgenefromorientalriverpawn(Macrobrachiumnipponense)SUNShengmingFUHongtuoGEXianpingZHUJianQIAOHuiJINShuboZHANGWenyiKeyLaboratoryofFreshwaterFisheriesandGermplasmResourcesUtiliz且tion,MinistryofAgriculture,FreshwaterFisheriesResearchCenter,Chines
5、eAcademyofFisherySciences;Abstract:TheY-boxproteinfamilyishighlyconservednucleicacidbindingproteinswhichareconservedfrombacteriatohuman.Inthisstudy,thecDNAencodingcoldshockproteinY~boxfromorientalriverpawnMacrobrachiumnipponense(MnY-box)wasclonedbyusingESTanalys
6、isandrapidampl辻icationofcDNAends(RACE)approach.ThefulllengthcDNAofMnY-boxwas1501bp,consistingofa5’untranslatedregionof84bp,a3’untranslatedregionof541bp,andanopenreadingframeof876bp.Thededucedproleinhad291aminoacidresidueswithmolecularmassof31.48kDa>Basedonthepro
7、teinsimilaritycomparison,theY-boxproteinfennilywasveryconservativeinCSDdomain.PhylogcnctictreeanalysisstatedthatorientalriverprawnhastheclosestrelationshipwithDaphniapulex.Quantitativereal-timeRT-PCRanalysisshowedthattheMnY-boxgenewasexpressedinhaemocytes,hepato
8、pancreas,muscles,gill,brain,testis,ovaryandintestineswiththehighestlevelofexpressioninthehepatopancreas.Afterenvironmentallowtemperatureandrecoverychallenge,therelati