全 文 ：Senescence-RelatedEndopeptidaseIsozymesin
SpirodelapolyrrhizaHalf-frondsDetectedbyGel
Electrophoresis
LIUQing-dai1 , WANGJin-ju1, 2 , LIHong-tao1, 2 , ZHAOYu1 , ZHANGZhi-zhou1, 3＊
1.ColegeofFoodEngineeringandBiotechnology, TianjinUniversityofScienceandTechnology, Tianjin300457;2.ColegeofLifeSci-
ences, NankaiUniversity, Tianjin300071;3.WeihaiCampus, HarbinInstiturteofTechnology, Weihai264209
Abstract　[ Objective] TostudyendopeptidasesinSpirodelapolyrhizahalf-frondsduringsenescenceandtheircharacters.[ Method] Changes
inendopeptidaseisoenzymesoftheSpirodelapolyrrhizahalf-frondsduringsenescenceweredetectedbygelatin-SDS-PAGEelectrophoresis,
andtheirtypeswereanalyzedwithproteaseinhibitors.[ Result] SixendopeptidasesweredetectedintheSpirodelapolyrrhizahalf-frondsduring
senescence.Amongthem, HEP1, HEP2, HEP4 andHEP6(highmolecular-weightendoprotease)weresenescence-relatedendopeptidases.
[Conclusion] Themetaloendopeptidaseplayssignificantrolesattheearlystageofsenescence, andthecysteineendopeptidasearethemost
abundantatthelatestageofsenescence.Keywords　Spirodelapolyrhiza;Half-fronds;Senescence;Endopeptidase
Received:November24, 2009　　Accepted:January15, 2010
SupportedbyNewCenturyExcelentTalentsProjectoftheMinistry
ofEducation(2006)andTalentsIntroductionFundofTianjinUni-
versityofScienceandTechnology(20060432 and20080216).
＊Corespondingauthor.E-mail:zhangzzbiox@gmail.com
　　Proteindegradationinvolvedbyproteaseisoneofthe
mostdistinctprocessesduringleafsenescence[ 1] .Theprote-
asesaredividedintotwotypes, endopeptidaseandexopepti-
dase, andtheformeraresupposedtoplayanimportantrole
duringleafsenescence[ 2].Duringsenescence, thetypesand
activityofproteaseinthesameplanttissuechangeevidently.
Therefore, toobservethechangesinactivityofendopepti-
daseisimportantforpreventingproteindegradationandap-
propriatelylengtheningphotosynthesis.Spirodelapolyrrhiza
half-frondsareleaveswithoutrootsandgrowingpoints.As
thesourceofcytokininisblocked, theSpirodelapolyrrhiza
half-frondissimilartotheexcisedleavesofadvancedplants
andthuscanbeusedasanexcelentmodelforstudyingse-
nescenceoftheexcisedleaf[ 3-4].Moreover, becausethe
lowersurfaceoftheSpirodelapolyrrhizahalf-frondscanwel
absorbnutrientsinmedium, theSpirodelapolyrrhizahalf-
frondcanbeusedtostudytheefectsofcompoundsdissolved
inmediumonsenescenceoffrondsinvitro.Previousreports
haveindicatedthatthesupplementationof6-benzyl-purine(6-
BA), anexogenouscytokinin, candelaysenescenceofhalf-
fronds[ 5] .Wedetectedthesenescence-relatedproteasesin
theSpirodelapolyrrhizahalf-frondsbythegelatin-SDS-PAGE
electrophoresis, estimatedtheirmolecularweightbychanging
theconcentrationofsubstrateproteininseparationgel, and
primarilyanalyzedtheirtypes.
MaterialsandMethods
Materials
Theshort-daymulti-rootP143 strain[ Spirodelapolyrhiza
(L.)Schleid, Lemnaceae] werecultivatedinlong-daylight(16
hlightand8 hdark)atthelightintensityof45 mol/(m2·s).
Themediawerechangedevery7d.Frondswerecrosscutin-
totwopieces, andhalf-frondswithoutrootsandsaceswere
culturedinsterilizedmedium.The6-BAwassupplementedat
aconcentrationof1 ×106 mol/L.
Extractionofcrudeenzyme
Atotalof0.5 gSpirodelapolyrrhizahalf-frondswasho-
mogenizedin3 mlTrisbufer(50mmol/L;pH7.5)andalit-
tlequartzsandinanice-bath, andthenthemixturewascen-
trifugedat1 000 r/minfor30 minat4 ℃.Thesupernatant
wasretainedandusedformeasuringproteincontentbythe
Bradfordmethodwithbovineserumalbuminasastandard.
Gelatin-SDS-PAGEgelelectrophoresis
Withbeta-mercaptoethanolfreesamplebufer, 75 g/L
separationgel, and40 g/Lconcentratedgeladdedgelatinat
aconcentrationof4 g/L, gelatin-SDS-PAGEwascarriedout
accordingtoliterature[ 6] .Aftertheelectrophoresis, thesep-
arationgelwasfulyimmersedintotherenatuarationbufer
(20 g/LTritonX-100, 50 mmol/LTrisbufer;pH7.5)for30
mintoeliminatetheSDS.Thenitwasincubatedin50 mmol/L
Trisbufer(pH7.5)at37 ℃ for8 handstainedbyamido
black.Afterdecolourization, whiteproteasebandsappeared.
EffectsofproteaseinhibitorsonactivityofSpirodela
polyrrhizaendopeptidase
Afterelectrophoresisandrenatuaration, theseparation
gelwascutinverticaldirection, andbeltswiththesamewidth
wereobtained.ThenPMSF(aserineendopeptidaseinhibi-
tor), EDTA(ametaloendopeptidaseinhibitor)andE-64 (a
cysteineendopeptidaseinhibitor)weresupplementedintothe
enzymereactionbuferrespectivelyataconcentrationof2
mmol/L, 2 mmol/Land25 μmol/L[1] .
ResultsandAnalysis
Changesinendopeptidaseisoenzymesinhalf-frondsdur-
ingsenescence
AscanbeseenfromFig.1, theactivityofendopeptidase
intheSpirodelapolyrrhizahalf-frondschangedgreatlyduring
senescence.Withaging, thehydrolyticactivityofendopepti-
dasewithgelatinassubstrategradualyincreased, andnew
endopeptidasesappeared.Becauseofthegreatermolecular
AgriculturalScience＆Technology, 2010, 11(1):31-32, 106
Copyright 2010, InformationInstituteofHAAS.Alrightsreserved. PlantPhysiologyandBiochemistry
DOI :10.16175/j.cnki.1009-4229.2010.01.002
weight, theseendopeptidaseswerenamedHEP1-6(highmo-
lecular-weightendoprotease)inaccordancewithmolecular
weight.OnlyoneproteasebandHEP3 appearedinthefresh
half-fronds, andatotalofsixproteasebandsappearedinthe
senescenthalf-fronds.TheHEP1appearedonDay3postse-
nescence, anditsactivitypeakedonDay5, begantodecline
sinceDay10 andlastonDay12.TheactivityoftheHEP2
peakedonDay8 andmaintainedatahighlevelatthelate
stageofsenescence.TheHEP3 showedactivityassoonas
senescenceappeared, anditsactivityalmostdidnotchange
withaging.TheHEP4 onlyappearedatthelatestageofse-
nescence, thatis, onDay10 andDay12.TheHEP5 ap-
pearedonDay3, anditsactivitychangedalittleduringse-
nescence.TheHEP6 alsoappearedatthelatestageofse-
nescence, thatis, onDay8, Day10 andDay12.
Inthelong-dayconditions, 6-BAdelayedthesenescence
oftheSpirodelapolyrrhizahalf-frondsandinhibitedtheex-
pressionofsenescence-relatedgenes.Therefore, theendo-
peptidasesregulatedbythe6-BAmaybecloselyrelatedto
thesenescenceofthehalf-fronds.Inthe6-BAtreatedhalf-
fronds, onlytheHEP1, HEP2, HEP5 andHEP6 appeared.
Furthermore, theactivityoftheHEP5 changedlitle, while
thatoftheHEP1, HEP2 andHEP6 greatlydeclined.The
HEP4 didnotappear.Therefore, theHEP1, HEP2, HEP4
andHEP6 maybesenescence-relatedendopeptidasesthat
welookedfor.
Determinationofmolecularweightofendopeptidase
Whentheconcentrationofgelatinwasreducedto0.5g/L,
thepositive-stainingstandardproteinbandsandthenegative-
stainingendopeptidasebandsbecameapparentatthesame
time(Fig.2).Fig.2 showsonlytwobandsinthegel, andthe
97-kDabandand66-kDabandhavealreadyrunoutthegel.
Accordingtomigrationrate, themolecularweightofthe
HEP1, HEP2, HEP4 andHEP5 wasrespectively120, 151,
181and196 kDa.
EfectsofproteaseinhibitorsonactivityofHEP1
Afterelectrophoresisandrenaturation, thepolyacrylam-
idegelswerecutintothebeltswiththesamesize.Theywere
separatelyincubatedintheenzymereactionbuferwithprote-
aseinhibitorsatthesameconcentration.Thegelbeltincuba-
tedintheenzymereactionbuferwithoutproteaseinhibitors
for5 dwasusedasacontrol.AscanbeseenfromFig.3
(thestainedbands), theactivityoftheHEP1 wasinhibitedby
E-64 butnotbyEDTAandPMSF.AndtheinhibitionofEDTA
wasmoreevident.Therefore, theHEP1 maybelongtothe
metaloendopeptidaseendopeptidase.Inaddition, theHEP2,
HEP4 andHEP6 wereinhibitedbyE-64;therefore, theymay
belongtothecysteineendopeptidases.
ConclusionsandDiscussion
Intheisozymespectrum, sixkindsofendopeptidaseap-
pearedintheSpirodelapolyrrhizahalf-frondsduringsenes-
cence.TheactivityoftheHEP1, HEP2, HEP4 andHEP6
wascontroledbycytokininandmayberelatedtosenes-
cence.Asshownbytheanalysiswithproteaseinhibitors, the
120-kDaHEP1 belongedtothemetaloendopeptidase, while
theHEP2, HEP4 andHEP6 belongedtothecysteineendo-
peptidase.
Themetaloendopeptidasesincludemanykindsofendo-
peptidasethatareresponsiblefordegradingextracelularma-
trixproteins, andthedegradationoftheextracelularmatrix
proteinsmaybeanimportantfactorcausingprogrammedcel
death(PCD).DelormeetalfoundthatCsl-MMP(amatrix
metaloproteinaserelatedtodegradationofmatrix)increases
whencotyledonofCucumissativusLinn.transitsfromsenes-
cencetoPCD, anditsfunctionsarethesameasthatofani-
malmatrixmetaloproteinases[ 7].TheHEP1 thatwasrelated
tothesenescenceofSpirodelapolyrrhizaappearedattheear-
lystageofsenescence(onDay3), anditsactivitywashigh-
eronDay5 andDay8.TheSpirodelapolyrrhizahalf-fronds
enteredirreversiblesenescenceafterDay8, possiblybecause
oftheinitiationofPCD.
Inmulticelulareukaryotes, PCDismediatedbydeathre-
ceptorincelsurfaceandinitiatedbyaseriesofcysteinepro-
tease[ 8] .TheHEP4 andHEP6 appearedatthelatestageof
senescence, andtheHEP1, HEP4 andHEP6 hadstrongac-
tivity.Theseresultsindicatedthatthecysteineendopeptidase
mayplayanimportantroleatthelatestageofleafsenes-
cenceandmaybecloselyrelatedtoPCD.
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丹参药渣中提取丹参酮的研究(摘要)
石岭 ,鱼红闪 ,金凤燮＊　(大连工业大学生物与食品工程学院 ,辽宁大连 116034)
[目的]水浸丹参和三七后药渣中提取丹参酮并确定丹参酮成分。
[方法]采用有机溶剂法提取丹参药渣中丹参酮 , 7种有机溶剂分别为甲醇、正己烷、石油醚、乙酸乙酯、丙酮、乙醚 、二氯甲烷 、三氯甲烷。以薄
层层析法检测确定最佳提取溶剂。在薄层层析板上 ,用毛细管吸取标准品及样品 ,点样于薄层层析板上 ,每次点样依照样品浓度确定点样次
数 ,每次点样均须用电吹风吹干。丹参酮展开剂的配方为:石油醚∶乙酸乙酯∶冰乙酸=8∶3∶1(V∶V∶V),直接观察;三七皂苷展开剂为V(氯
仿)∶V(甲醇)∶V(水)=14∶6∶1,用 10%H2SO4 加热显色。通过高效液相色谱法确定丹参药渣中丹参酮成分。高效液相色谱法(HPLC):高效
液相色谱仪为美国Waters2695-2996DAD;色谱柱为HypersilODS2 5μm(4.6mm×150.0mm)fromElite;进样量 , 10μl;体积流量 , 1.0ml/min。
洗脱液A为水 , B为甲醇。洗脱条件 ,初始时 , A为 40%, B为 60%;5min时 , A为 40%, B为 60%;20min时 , A为 20%, B为 80%;30min时 ,
A为 20%, B为 80%。
[结果]乙醚为丹参药渣中提取丹参酮的最佳溶剂。水浸丹参和三七后干药渣 ,先经乙醇提取得到脂溶性提取物 , 再以乙醚为溶剂进行索氏
提取 ,丹参酮混合物得率为 2.17%;高效液相色谱法检测 ,丹参酮混合物中丹参酮ⅡA、次甲丹参醌、隐丹参酮、丹参酮I含量分别为 3.62%、
1.02%、2.56%、2.75%。
[结论]丹参药渣中丹参酮成分与药材丹参基本一致 ,丹参药渣可以作为一种丹参酮资源 ,具有开发利用的价值。
关键词　丹参药渣;丹参酮;提取
基金项目　国家自然科学基金资助项目(30470055);辽宁省教育厅创新团队项目(2007T006)。
作者简介　石岭(1983-),男,河南汝南人 ,硕士研究生,研究方向:天然产物中活性物质的研究。 ＊通讯作者。
收稿日期　2009-11-11　　修回日期　2010-01-20
(上接第 32页)
用凝胶电泳法研究紫萍半叶状体衰老期间的内肽酶同工酶(摘要)
刘清岱 1 ,王金菊 1, 2 ,李红涛 1, 2 ,赵 昱 1 ,张治州 1, 3＊　(1.天津科技大学食品工程与生物技术学院 ,天津 300457;2.南开大学生
命科学学院 ,天津 300071;3.哈尔滨工业大学威海分校 ,山东威海 264209)
[目的]研究与紫萍半叶状体衰老相关的内肽酶及其性质。
[方法]采用明胶-SDS-聚丙烯酰胺凝胶电泳技术分析与衰老相关的内肽酶同工酶。通过改变电泳分离胶中底物蛋白浓度 ,可以使正染的蛋
白质标准条带和负染的内肽酶条带同时显现出来 ,从而了解内肽酶的表征分子量。将进行电泳 、复性后的聚丙烯酰胺凝胶切成相同大小的
条带 ,分别置于含有同等浓度蛋白酶抑制剂[ 2mmol/L丝氨酸型内肽酶抑制剂(PMSF)、2mmol/L金属型内肽酶抑制剂(EDTA)、25μmol/L
半胱氨酸型内肽酶抑制剂(E-64)]的酶反应缓冲液中进行反应 ,同时以不加抑制剂的胶条处理 5d的样品作为对照 ,以分析内肽酶类型。
[结果]衰老紫萍半叶状体的内肽酶同工酶谱上共出现 6种内肽酶。其中 ,内肽酶HEP1、HEP2、HEP4和 HEP6在衰老过程中出现 ,且活性受
细胞分裂素的调控 ,可能是衰老相关的内肽酶。经过蛋白酶抑制剂的分析结果表明 ,与对照相比 , E-64未能对内肽酶条带 HEP1产生抑制作
用 ,而EDTA和PMSF都能抑制内肽酶活性 ,且EDTA作用较明显 ,因此 ,初步鉴定分子量为 120kD的 HEP1为金属型内肽酶 ,其他 3种内肽
酶均为半胱氨酸型内肽酶。
[结论] 在紫萍半叶状体衰老早期金属型内肽酶活性较强 ,而在衰老后期半胱氨酸型内肽酶大量出现并具有较高的活性。
关键词　紫萍;半叶状体;衰老;内肽酶
基金项目　教育部新世纪优秀人才计划(2006);天津科技大学人才引进基金(20060432, 20080216)。
作者简介　刘清岱(1975-),男 ,河北保定人 ,博士 ,讲师,从事植物生理与分子生物学研究。＊通讯作者。
收稿日期　2009-11-24　　修回日期　2010-01-15
106 AgriculturalScience＆TechnologyVol.11, No.1, 2010