龙眼SPFH家族全基因组鉴定进化分析与功能预测

功能预测1福建农林大学园艺植物生物工程研究所福州3500022山西农业大学经济作物研究所汾阳032200摘要SPFH(Stomatin,Prohibitin,FlotillinandHflk/C)是一种膜微区蛋白，该家族的蛋白均含有一个高SPFH基于龙眼基因组数据库，对龙眼SPFH(DlSPFH)进行家DlSPFH包含DlSlp(1个)、DlElp(1个)、DlFlots(2个)、DlPHBs 员。基因结构与保守基序分析结果显示，DlSPFH各成员间基因结构存在较大的差异；基序以motif1最为HsHGenome-wideidentification,evolutionaryanalysisandfunctionalimocarpuslonganLourZHANGChunyu1#,ZHANGZihao1#,CHENYukun1,LINYuling1,CHENZhenguang1,WANGJing1,2&LAIZhongxiong1**1InstituteofHorticulturalBioengineering,FujianAgricultureandForestryUniversity,Fuzhou350002,China2InstituteofEconomicCrops,ShanxiAgriculturalUniversity,Fenyang032200,ChinaAbstractSPFH(Stomatin,Probitin,FlotillinandHFLK/C)isamembranemicrodomainproteinthatishighlyconserved.Inplants,theSPFHfamilyproteinscanbesubdividedintofivesubfamilies:SLP(stomatin-likeprotein),PHB(Flotillin),FLOT(Flotillin),ELP(Erlin-likeprotein)andHIR(Hypersensitiveinducedreaction).Atpresent,therearefewreportsonthegenome-wideidentificationofSPFHinplants,andonlyafewsubfamiliesofSPFHhavebeenstudiedinplantssuchasArabidopsisthalianaandrice.Therefore,inordertounderstandthemolecularcharacteristicsofmembranemicrodomainproteinSPFHinlongan.Basedonthelongangenomedatabase,thefamilymemberidentificationandevolutionanalysisoflonganSPFH(DlSPFH)wasinvestigated,andcombinedwiththelongan收稿日期Received:2020-10-25接收日期Accepted:2021-01-07国家自然科学基金项目(31572088)；福建省高原学科建设经费(102/71201801101)；福建农林大学科技创新专项基金(KFA19037A)资助SupportedbytheNationalNaturalScienceFoundationofChina(31572088);FundsforplateauDisciplineConstructioninFujianProvince(102/71201801101);SpecialFundforScienceandTechnologyInnovationofFujianAgricultureandForestryUniversity(KFA19037A)#并列第一作者Co-firstauthors**通讯作者Correspondingauthor(E-mail:Laizx01@163.com)transcriptomedatabasetoanalyzetheexpressionpatternsofDlSPFHsduringearlysomaticembryogenesis.TheresultsshowedthattheDlSPFHfamilycontained14members,including5subfamilies:DlSlp(1),DlElp(1),DlFlots(2),DlPHBs(2)andDlHIRs(8);Thenumberofaminoacids,molecularweightandisoelectricpointofthefamilymemberswerebetween193-2570aa,21.49-282.11KD,5.38-9.42respectively,andtheylocatedon8chromosomes.ThereweretwopairsoftandemduplicationgenesandthreepairsoffragmentduplicationgenesintheDlSPFHsfamily.Inaddition,therewere17,14,and2pairsofcollinearitymembersinDlSPFHsandArabidopsis,sweetorange,riceSPFHfamilymembers,respectively.TheanalysisofgenestructureandconservedmotifsshowedthatthereweresignificantdifferencesingenestructureamongthemembersofDlSPFHandmotif1wasthemostconservedmotif.TheproteininteractionpretectionresultsshowedthatDlSPFHsnotonlyinteractedwithfamilymembers,butalsointeractedwithavarietyofproteins.DlSPFHsincludedmanyhormoneresponseelementsinthepromoterregion,andtheymightbepreciselyregulatedby11miRNAs.Intheearlystagesofsomaticembryogenesis,DlHIR1,DlHIR2,DlHIR3,DlPHB1,DlHIR8showedhigherexpressionlevelsofFPKMthanothermembers.StudieshadshownthatthegeneduplicationeventsofDlSPFHsweremainlyfragmentduplication,andmightbeinvolvedindiseaseresistance,hormoneresponse,andsomaticembryogenesisinlongan.KeywordsLongan;SPFH;Genome-wideidentification;evolutionaryanalysis;functionalprediction筏模型[2]。脂筏是富含胆固醇和鞘磷脂的一种动态结构域，同时也是细胞表面具有特定结构和功能的质膜微区[3]。膜微区(membranemicrodomains)因其特殊的结构特点，能够促进蛋白之间、蛋白与脂类之间的相互作用，并且参与膜的信号传导、物质跨膜运输以及病原微生物的侵染等过程，故对植物的抗病以及生的作用[4-5]。SPFH(Stomatin,Prohibitin,FlotillinandHflk/C)蛋白属于膜微区的一类蛋白。在植物中，SPFH家族tinbulumlipidraftassociatedprotein 达，还能够响应病原体、非生物胁迫以及激素传导过程[6]。HIR是anypersensitiveresponseLRRLeucinerichrepeat导细胞凋亡过程以及对细菌断制约着果实的品质以及产量。此外，龙眼的胚胎发育情况也对其果实品质以及产量起着至关重要的作用BR达情况的探析，以期探索龙眼SPFH的潜在生物学功能，为后续龙眼的抗病机制以及胚胎发育的进一步研1.1材料本研究基于实验室构建的第三代龙眼基因组、龙眼转录组数据库进行分析；拟南芥(Arabidopsisthaliana)、水稻(Oryzasativa)、番木瓜(Caricapapaya)以及甜橙(Citrussinensis)的SPFH氨基酸序列于phytozome12(/pz/portal.html)获取。共线性分析所需拟南芥、水稻以及甜(Citrussinensis)GenomeAnnotationProject网站(/orange/download/index.php)。1.2DlSPFH家族成员鉴定与蛋白序列分析等电点(pI)、不稳定系数以及亲水性进行预测分析；信号肽的预测采用SignalP4.1；采用NBA-Palm (/bioinf/plant-multi/#)对DlSPFH蛋白进行亚细胞定位预测。SPFH家族成员系统进化分析树的构建。首先，采用MUSCLE对序列进行比对；其次，设置参数NJ法(Neighbor-joiningmethod)、1.4DlSPFH家族染色体定位与共线性分析1.5DlSPFH家族成员基因结构以及保守基序motif分析基于龙眼基因组gff注释文件，利用TBtools将DlSPFH成员的基因结构进行可视化；利用MEME1.6DlSPFH家族成员顺式作用元件分析采用TBtools提取DlSPFH家族成员CDS上游2000bp的序列，将其提交至PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)在线网站进行启动子顺式作用元件的预测，最后采用TBtools将结果可视化。1.7DlSPFH家族成员蛋白互作预测1.8DlSPFH家族成员miRNA预测tpplantgrnnobleorgpsRNATarget1.9DlSPFH家族成员早期体胚发生过程的表达分析实胚性结构(Incompletecompactpro-embrogeniccultures,ICpEC)以及球形胚(Globularembryos,GE)结果与分析2.1DlSPFH家族成员鉴定与蛋白序列分析lSPFH表1DlSPFH家族成员蛋白理化性质分析Table1AnalysisofphysicochemicalpropertiesofDlSPFHfamilymembers基因ID基因名基因位置氨基酸umberKDlecularoweightpI不稳定系平均亲水性数Hydrophilicitybility棕榈酰化位信号肽点个数跨膜结构域00线粒体DlElp101线粒体DlFlot140细胞核DlFlot210细胞核B00线粒体B00线粒体DlHIR120线粒体DlHIR210线粒体DlHIR310线粒体DlHIR401细胞核DlHIR520线粒体DlHIR610线粒体10线粒体10线粒体SPFH析Slp以及HIR5个亚组。其中龙眼Flot共有2个成员，分别为DlFlot1-DlFlot2；PHB共有2个成员：图1龙眼、拟南芥、水稻、番木瓜、甜橙SPFH家族成员系统进化分析.Fig.1PhylogeneticanalysisofSPFHfamilymembersinlongan,Arabidopsis,rice,papayaandsweetorange.2.3DlSPFH家族成员染色体定位分析图2DlSPFH家族成员染色体定位分析。带*为DlSPFH家族串联重复基因，使用红色线条相连的基因为片段重复基因对.Fig.2ChromosomelocationanalysisofDlSPFHfamilymembers.Thesymbol*indicatedthetandemduplicationgenesofDlSPFHfamily,andthegenesconnectedbytheredlinewerethefragmentrepeatgenepairs.2.4龙眼与拟南芥、水稻、甜橙SPFH家族成员共线性分析MCScanX南芥、水稻、甜橙进行共线性分析，结果显示：龙眼与拟南芥SPFH家族成员共存在17对共线性成员(图3A)，与水稻SPFH家族成员仅存在2对共线性成员(图3B)，与同为木本植物的甜橙SPFH家族成员具有14对共线性基因对(图3C)。对这些共线性成员的进化选择压力进图3龙眼与拟南芥(A)、水稻(B)、甜橙(C)SPFH家族成员共线性分析。Fig.3CollinearityanalysisofSPFHfamilymembersinlonganandArabidopsis(A),longanandrice(B),longanandsweetorange(C).表2龙眼与拟南芥、水稻、甜橙SPFH家族共线性成员进化选择压力分析Table2Analysisofevolutionaryselectionpressureonco-linearmembersofSPFHfamilyinlonganandArabidopsis,longanandrice,longanandsweetorange.种类各物种成员龙眼成员拟南芥AtFlot1DlFlot1DlElp1BBBBBBBDlHIR6DlHIR8DlHIR8DlHIR1DlHIR1DlHIR1DlHIR2水稻DlElp1DlHIR2DlHIR2DlHIR1DlElp1DlHIR6DlHIR8DlFlot1甜橙DlHIR1DlHIR6DlHIR8DlHIR1DlHIR6DlHIR8B2.5DlSPFH家族成员基因结构与保守基序motif分析个成员(DlHIR2、DlHIR6、DlHIR7)包含5个外显子，4个内含子；2个成员(DlHIR1、DlHIR8)包含6图4DlSPFH家族成员基因结构位置(A)以及保守基序motif(B)分析.Fig.4Analysisofgenestructurelocation(A)andconservedmotif(B)ofDlSPFHfamily.2.6DlSPFH成员启动子顺式作用元件分析的核心启动子元件(CAAT-box、TATA-box)以及光响应元件，说明它们都具有响应光的能力；此外，分p图5DlSPFH家族成员启动子顺式作用元件.Fig.5Cis-actingelementsofDlSPFHfamilymembers.2.7DlSPFH成员蛋白互作分析家族以外，其余亚家族成员均能够与EF-TU蛋白(AT4G02930)发生互作。图6DlSPFH家族成员蛋白-蛋白互作网络Fig.6Theprotein-proteininteractionnetworkofDlSPFHfamilymembers2.8DlSPFH家族成员受到靶向调控的miRNA种类分析E表3DlSPFH家族成员受到靶向调控的miRNA种类Table3SpeciesofmiRNAstargetedofDlSPFHfamilymembers基因名miRNA(E)DlFlotmiR440(3.0)DlHIRmiR406(4.5)DlHIR4DlHIR5DlHIRmiR774b(4.5)DlHIRmiR774b(4.5)2.9DlSPFH家族成员体胚发生早期表达分析Fig.7ExpressionanalysisofDlSPFHfamilymembersduringearlyembryogenesisEC:Embryoniccallus,ICpEC:Incompletecompactpro-embrogeniccultures,GE:Globularembryos.3讨论3.1DlSPFH家族成员鉴定与进化特性尽管目前在拟南芥中鉴定出了Elp(1个)，Slp(2个)，PHB(7个)，Flot(3个)，HIR(4个)RHBresponse果显示，DlPHB成员都定位于线粒体中，表明其可能参与龙眼SPFH植物还未分化前就已经3.2DlFlots与DlHIRs可能参与龙眼的抗病机制JAGmHIR1可能通过调控细胞周期及细3.3DlSPFHs可能参与龙眼体胚发生过程3.4DlElp1可能具有功能多样性ina4结论 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