全 文 ：AFLP Analysis ofPhylogenetic Relationship of
Salviaspp.inBeijing
YANGJian-yu, CHENHong-wei, LIUKe-feng＊ , WANGHong-li, LIUYong-guang, WANGShun-li, JIN
Zhu-li-da
SalviaProjectTeam, BeijingUniversityofAgriculture, Beijing102206
Abstract Twenty-thirdcolectionsofSalviaspp.inBeijingwereanalyzedphylogeneticalybyAFLPtechnique.6 primerpairswithgoodrepeti-
tionandhighlypolymorphicbandsfrom 21pairswereselectedtoamplifythegenomicDNA.Alof367AFLPbandswereobtained, inwhich359
(97.82%)werepolymorphicmarkers.Atageneticdistanceof0.32, thecolectionscouldbeclusteredinto7speciesi.e.S.splendens, S.fari-
nacea, S.coccinea, S.plebeia, S.miltiorhiza, S.umbraticaandS.oficinalis.TherespectivegeneticrelationshipbetweenS.farinacea, S.coccinea, S.plebeia, S.miltiorhiza, S.umbratica, S.oficinalisandS.splendenswerefurtherorderbyorder.Therespectivegeneticrelation-
shipbetweencultivarsandcommercialvarietiesofS.splendenswasfurther.Theresultsprovidedreferenceforhybridbreedingbetweeninside
andoutsideofspeciesofSalviaspp.inBeijing.
Keywords Salviaspp.;AFLP;Geneticsanalysis;Salviasplendens
Received:December21, 2009 Accepted:February3, 2010
SupportedbyBeijingMunicipalCommissionofEducationProject
(KM200910020014).
＊Correspondingauthor.E-mail:liukefeng006@163.com
TheSalviaspp.isinLabiataefamilyandithasannual
herbs, biennialherbs, evergreenperennialherbs, subshrub
andshrubs.Therearemorethan1 000 speciesintheworld
whicharemainlydistributedintropicandtemperatezone[ 1] .
Thereareabout9 variants, 25 varietiesand83 speciesinthe
world, whiletheyaremostlyinSouthwestChina[ 2] .Thecom-
monsamegenericplantsinBeijingareaareS.splendens, S.
coccinea, S.farinacea, S.oficinalis, S.umbratica, S.milti-
orrhizaandS.plebeiaetal.Withtheincreaseofdevelopment
andutilizationlevelofnaturalresources, theplantsinSalvia
spp.arewidelyusedinmedicine, food, perfume, cosmetic
industryandornamentalfield[ 3] .Forexample, S.officinalis
wasusedassafeandbalmyvegetableswithhighK, Mg, Fe
andlowNainEuropeandAmerica[ 4] .Sotheyhavebrightfu-
tureandhigheconomicvalue.
TheplantsinSalviaspp.aredistributedwidelyandthey
havemanygreatvarieties, sotherearecrosspolinationphe-
nomena[ 5] .Thevariationsarebigandthegeneticrelation-
shipsarecomplex, whilethesystematicclassificationandcul-
tivaridentificationandresourceutilizationofplantsinthisgen-
eraareextremelyconfused[ 6] .KhaliletalhaveusedRAPDto
analyzegeneticrelationshipsofthreekindsofplantsinSalvia
spp.[7] ;TIANYe-linetalhaveusedRAPDmolecularmarker
techniquetoanalyzeDNApolymorphismsofeightkindsofS.
splendens[ 8] ;GUOBao-linetalhaveconductedRAPDanaly-
sisofcommunalgeneticrelationshipofS.miltiorrhiza[ 9] .
However, thestudiesaboutgeneticrelationshipofplantsin
Salviaspp.arefew, besides, thepreviousstudiesonlyana-
lyzeoneorfewspeciesinSalviaspp.andthestudiesonin-
terspecificrelationshipinSalviaspp.throughAFLParerepor-
tedseldomly.
AFLPtechnologywasusedinthisexperimenttoanalyze
thegeneticrelationshipof23 kindsofplantsinSalviaspp.
whichwere7 speciesforrevealingthegeneticrelationshipbe-
tweendominantspeciesanddominantcommercialspecies.
Thisexperimentprovidedtheoryreferenceforcarryingout
widecrossbreedinginintraspeciesandamonginterspeciesof
plantsinSalviaspp.inBeijingareaandreducedblindnessof
breedingwork.
MaterialsandMethods
Experimentalmaterials
Theexperimentalmaterialscontained7 kindsofplantin
Salviaspp.inBeijingarea, whichwereS.splendens(self
bredinbredlinesof9 projectteamand8 commercialspecies
), S.farinacea, S.coccinea, S.plebeia, S.miltiorrhiza, S.
umbraticaandS.oficinalis.Theconcretematerilaswerelis-
tedinTable1.
Table1 Thespeciesname, varietynameandflowercoloroftest
materials
No. Speciesname Varietyname Flowercolor
1 S.splendens BN5(selfbredinbredline) Red
2 S.splendens BN15(selfbredinbredline) Red
3 S.splendens BN23(selfbredinbredline) Purepink
4 S.splendens BN30(selfbredinbredline) Red
5 S.splendens BN47(selfbredinbredline) Red
6 S.splendens BN67(selfbredinbredline) White
7 S.splendens BN72(selfbredinbredline) Orange
8 S.splendens BN35-1(selfbredinbredline) Red
9 S.splendens BN35(selfbredinbredline) Red
10 S.splendens VistaLavender lavender
11 S.splendens Salsa Red
12 S.splendens Desert Red
18 S.splendens ScarletKing Red
19 S.splendens ScarletQueen Red
20 S.splendens RedHotSaly Red
21 S.splendens BlazeofFireSeriesRed Red
23 S.splendens OlympicFlame Red
13 S.coccinea Red
14 S.umbratica Blue
15 S.farinacea Blue
16 S.oficinalis Blue
17 S.miltiorrhiza Blue
22 S.plebeia Blue
AgriculturalBiotechnologyAgriculturalScience＆Technology, 2010, 11(2):72-75Copyright 2010, InformationInstituteofHAAS.Alrightsreserved.
DOI :10.16175/j.cnki.1009-4229.2010.02.006
Experimentalmethod
DNAextraction Thetenderleavesof10 to15 plantsinev-
eryspecieswerecutintopiecesandmixed, thentheywere
groundinliquidnitrogenquickly.Afterground, theywereput
into2mlcentrifugetubeandpreservedat-70 ℃ forfuture
use.TheplantGenomicDNAkitwasusedtoextractDNA
fromsamplesaccordingtoinstructionofkit.TheDNAwere
conducted0.8% agarosegelelectrophoresisexperiment,
thenthesampleconcentrationswereadjustedto50 ng/μlfor
futureuseafterdetection.
AFLPanalysis TheAFLPamplificationwasinaccordance
withmodifiedVossmethod[10] .TheEcoRⅠ andMseⅠ restric-
tionendonucleasewereusedtoconductdoubleendonucle-
ase, meanwhilerestrictionfragmentswereconnectedwith
jointsofEcoRⅠ andMseⅠ(Table2)underT4DNALigaseefect, thentheenzymereactionwaslastedfor12 h.There-
actionsolutionafterrestrictionenzymeconnectionwerediluted
6 timesaspre-amplificationtemplateandthepre-amplifiedre-
actionconditionswerelistedas94 ℃, 30 s;56 ℃, 60 s;72
℃.60s, 30 cycles(BIORAD:MyCyclerthermalcyclertype
PCRinstrument, thesameasbelow).Thepre-amplified
productsweretakenasPCRreactionmodelofselectiveam-
plificationafterdiluted20 times.Theselectiveamplification
primerswere6 pairsofprimerwithhighpolymorphismwhich
werechosenfrom21 pairsofprimerinpre-test(E+ATT/M+
AC;E+ATT/M+GA;E+AAG/M+CTG;E+AAG/M+
CCT;E+AAG/M+ACT;E+AAC/M+ACT).Thereaction
systemwasdividedintotwosteps:①gradientcoolingamplifi-
cation.Thefirstcyclewas94 ℃, 30 s;65 ℃, 30 s;72 ℃,
60s.Theannealingtemperatureoffolowingeverycyclewas
declined0.7 ℃successivelyandthereweretotaly13 cycles.
②Commonamplification.94 ℃, 30 s;56 ℃, 30 s;72 ℃, 60
s;23 cycles.Afteramplification, theamplifiedproductswere
conductedelectrophoresison6% polyacrylamidegelinDYG-
20Aelectrophoresistankat60W ofBG-Power3 500 power
supplyuntilbromophenolblueindexlinewasoutofglass
plate.
Table2 AdaptersandprimerssequenceusedinAFLPanalysis
Adaptersandprimers Sequence(5′※ 3′)
EcoRⅠadapter CTCGTAGACTGCGTACCAATTGG-TACGCAGTC
MseⅠadapter GACGATGAGTCCTGAGTACTCAG-GACTCAT
EcoRⅠ-00 GACTGCGTACCAATTC
MseⅠ-00 GATGAGTCCTGAGTAA
E+ATT/M+AC GACTGCGTACCAATTCATT/GAT-GAGTCCTGAGTAAAC
E+ATT/M+GA GACTGCGTACCAATTCATT/GAT-GAGTCCTGAGTAAGA
E+AAG/M+CTG GACTGCGTACCAATTCAAG/GAT-GAGTCCTGAGTAACTG
E+AAG/M+CCT GACTGCGTACCAATTCAAG/GAT-GAGTCCTGAGTAACCT
E+AAG/M+ACT GACTGCGTACCAATTCAAG/GAT-GAGTCCTGAGTAAACT
E+AAC/M+ACT GACTGCGTACCAATTCAAC/GAT-GAGTCCTGAGTAAACT
Dataanalysis Afterstainedbysilver, theamplifiedbands
wererecordedunderlighttoform binarymatrix.TheNT-
SYSpc2.10esoftwarewasusedforanalysisandUPGMA
methodwasusedtoconductclusteranalysis, whichformed
clustergraphthroughTreeplotmodule.
ResultsandAnaysis
AFLPamplifiedresultsandpolymorphismanalysis
Thescreened6 pairsofprimerswereconductedAFLP
analysisto23 kindsofplantsinSalviaspp.(Fig.1), through
thiskindofanalysis, therewere367 amplificationsites, which
had359 polymorphicloci, taking97.82% oftotalamplification
sites.
1-23:Samples1-23;M:Marker.
Fig.1 AFLPelectrophoretogramof23samplesofSalviaspp.ampli-
fiedbyE+ATT/M+AC
Geneticdistanceandclusteranalysis
NTSYSpc2.10esoftwarewasusedtoconductanalysis
andUPGMAwasusedforclusteranalysistoobtaingenetic
distancematrix.Thegeneticdistancesbetweeneverytwoex-
perimentalmaterialswerefrom0.004 8 to0.840 8, thenUP-
GMAmethodwasusedforclusteranalysistoobtaincluster
tree(Fig.2).AccordingtoclusterresultofFig.2, thecombi-
ninglinewasdrawnat0.32 geneticdistance, whichcoulddi-
vide23 kindsofplantsinSalviaspp.into7groups:S.splen-
densgroup, S.farinaceagroup, S.coccineagroup, S.ple-
beiagroup, S.miltiorrhizagroup, S.umbraticagroupandS.
officinalisgroup.Among7speciesofSalviaspp.Therelation
betweenS.farinaceagroupandS.splendensgroupwas
close, whiletherelationsamongS.coccinea, S.plebeia, S.
miltiorrhiza, S.umbratica, S.officinalisandS.splendens
werefromfartoclose.ThegeneticdistancesbetweenS.
coccinea, S.plebeia, S.miltiorrhiza, S.umbratica, S.ofici-
73YANGJian-yuetal.AFLPAnalysisofPhylogeneticRelationshipofSalviaspp.inBeijing
nalisandS.farinaceawerefromsmaltobig, whilethege-
neticdistancesbetweenS.plebeia, S.miltiorrhiza, S.um-
bratica, S.oficinalisandS.coccineawerefromsmaltobig.
ThegeneticdistancesbetweenS.miltiorrhiza, S.umbratica,
S.officinalisandS.plebeianwerefromsmaltobig, whilethe
geneticdistancesbetweenS.umbraticaandS.oficinaliswas
increasedfromsmaltobig.TherelationsbetweenS.splen-
dens, S.plebeia, S.miltiorrhiza, S.umbraticaandS.coc-
cineawerefar.
Fig.2 Dendrogramof23samplesofSalviaspp.
InS.splendens, whenthecoefficientofgeneticdistance
was0.06, theexperimentalsamplesweredividedinto6
groups.TheBN5, BN47, BN23, BN30, BN15, BN72, BN67,
BN35-1 andBN35wereconductedclusteranalysisonegroup;
VistaLavenderwasthesecondgroup;ScarletKing, Scarlet
Queen, RedHotSaly, BlazeofFireSeriesRedwerethe
thirdgroup;theOlympicFlamewasfourthgroup;Desertwas
fifthgroupandSalsawasthesixthgroup.Inthecommercial
S.splendensspecies, therelationbetweenSalsaandother
S.splendensspecieswerefar, whileBN5 andBN47, BN15
andBN67, BN35-1 andBN35 werefirstlyclustered, sotheir
geneticrelationshipwereclose.
ConclusionsandDiscussions
AFLPmolecularmarkertechniqueisusedtoanalyze23
kindsofplantsinSalviaspp.inBeijingarea, then367 amplifi-
cationsiteswhichhave359 polymorphicloci, taking97.82%
oftotalamplificationsites.Whenthecoefficientofgeneticdis-
tancewas0.32, thesamplesaredividedinto7 groups:S.
splendensgroup, S.farinaceagroup, S.coccineagroup, S.
plebeiagroup, S.miltiorrhizagroup, S.umbraticagroupand
S.officinalisgroup.Theanalysisresultsshowthatplantmate-
rialsarein7speciesofSalviaspp., soAFLPmolecularmark-
ertechniqueissuitableforphylogeneticanalysisofplantsin
Salviaspp..
Theresearchresultsdemonstratethattherelationsbe-
tweenS.splendensandS.farinacea, S.coccinea, S.ple-
beia, S.miltiorrhiza, S.umbratica, S.oficinalisarefrom
closetofar, whileinS.splendensgroup, therelationsbe-
tweenself-chosenspeciesandcommercialspeciesisfar.In
geneticbreeding, theplanthybridizationinvariety, strains
withfargeneticrelationshiporevenininterspeciesandinter-
generacanculturehybridswithstronggrowthrateandmeta-
bolicfunction, sotheyhavedevelopedorgans, bigbodytype,
highyieldorhavehighantiviralability, insectresistanceca-
pacity, anti-adversityability, viability, fecundityandsurvivabil-
ityetal.DuringthedistanthybridizationbreedingofS.splen-
dens, theparentscrosscombinationcanbechosenselective-
lyaccordingtheexperimentalresults.Inordertoenhance
stressresistance, theinterspecifichybridizationingeneracan
beconductedandS.oficinalisistakenasoneparent;ifthe
hybridizationcompatibilityisconsideredandthesuccessful
rateisincreasedatthesametime, S.farinaceaorS.coc-
cineawithcloserelationtoS.splendensshouldbetakenas
oneparent.Forbreedingwork, inordertoobtainofspring
withgoodtraits, thecrosscombinationofselfbredinbred
speciesand commercialspecies should be givenmore
emphasis.
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Responsibleeditor:ZHANG Cai-li Responsibletranslator:LIZhu-le Responsibleproofreader:WU Xiao-yan
北京地区常见鼠尾草属植物 AFLP亲缘关系分析(摘要)
杨建玉 ,陈洪伟 ,刘克锋＊ ,王红利 ,刘永光 ,王顺利 ,金珠理达 (北京农学院一串红项目组 ,北京 102206)
[目的 ]采用AFLP分子标记技术对北京地区鼠尾草属(Salviaspp.)植物的 7个种 23份样本进行亲缘关系分析。
[方法 ]利用 21对引物组合中筛选出的稳定性好、多态性较高的 6对引物(E+ATT/M+AC;E+ATT/M+GA;E+AAG/M+CTG;E+AAG/M
+CCT;E+AAG/M+ACT;E+AAC/M+ACT)用于扩增北京地区鼠尾草属植物基因组DNA,反应体系分为两步:①梯度降温扩增。第 1个循
环为 94℃, 30s;65℃, 30s;72℃, 60s。以后每个循环的退火温度依次降低 0.7℃,共 13个循环;②普通扩增。 94℃, 30s;56℃, 30s;72℃,
60s;23个循环。扩增后采用 6%聚丙烯酰胺凝胶和BG-Power3500型电源和DYC-20A型电泳槽 ,在恒功率 60 W的条件下电泳至溴酚蓝指
示线刚刚跑出玻璃板。银染后在灯光下统计扩增条带数 ,构成二进制矩阵表。用 NTSYSpc2.10e软件进行分析 ,采用 UPGMA方法进行聚类
分析 ,通过Treeplot模块生成聚类图。
[结果 ]对 23份鼠尾草属植物进行 AFLP分析 ,得到 367个扩增位点 ,其中多态性位点 359个 ,占总扩增位点的 97.82%。在遗传距离为 0.32
的水平下 , 23份鼠尾草属植物样本归为 7个组:一串红(S.splendens)组、蓝花鼠尾草(S.farinacea)组、朱唇(S.coccinea)组 、雪见草(S.plebe-
ia)组 、丹参(S.miltiorrhiza)组 、荫生鼠尾草(S.umbratica)组和罗马尼亚鼠尾草(S.oficinalis)组。蓝花鼠尾草、朱唇、雪见草、丹参、荫生鼠尾
草 、罗马尼亚鼠尾草与一串红的亲缘关系依次增远;一串红种内 ,自选品种(系)与商品种亲缘关系较远。
[结论 ]该研究结果可为北京地区开展鼠尾草属植物的种间和种内远缘杂交提供理论参考。
关键词 鼠尾草属;AFLP;亲缘关系;一串红
基金项目 北京市教委 “北京地区草花引种研究及标准化生产规程建立 ”课题项目(KM200910020014)。
作者简介 杨建玉(1984 -),女 ,北京人 ,硕士研究生 ,研究方向:一串红育种与生理。 ＊通讯作者。
收稿日期 2009-12-21 修回日期 2010-02-03
(上接第 14页)
HpaGXoo蛋白的结构与功能分析(摘要)
任秀艳＊ ,冯雪 ,侯志敏 ,张倩倩 (廊坊师范学院生命科学学院 ,河北廊坊 065000)
[目的 ]分析HpaGXoo蛋白的结构与功能 ,为研究两者的关系提供理论依据。[方法 ]利用ISREC(htp://www.isrec.isb-sib.ch/software/SAPS form.html)中提供的工具SAPS进行分子量、分子式 、等电点、氨基酸所占比
例 、带电荷情况等参数的在线分析 ,并利用瑞士生物信息学研究所提供的ProtParam(htp://us.expasy.org/cgi-bin/protparam)程序进行氨基
酸残基数目 、组成、蛋白质相对分子质量、理论等电点及平均可塑性、疏水性等参数的分析;利用NPSA(htp://npsa-pbil.ibcp.fr)中的 MLRC
程序在线分析α-螺旋、无规则卷曲以及延伸链等的预测;利用 TMHMM 2.0Server(htp://www.cbs.dtu.dk/services/TMHMM/)和 TMpred
(htp://www.ch.embnet.org/software/TMPRED form.html)2个软件同时对HpaGXoo蛋白序列的跨膜区域进行分析 ,用 SignalP(htp://www.
cbs.dtu.dk/services/SignalP/)服务器预测HpaGXoo的信号肽;用PSORTWWWServer(htp://psort.nibb.ac.jp/)中的PSORTPrediction工具进行细胞定位;利用网站 Expasy(htp://au.expasy.org/tools/)中 SwissModel程序可同源建模 , 推测 HpaGXoo蛋白的三维结构或用Phyre(前身
3D-PSSM)(htp://www.sbg.bio.ic.ac.uk/～phyre/)软件预测蛋白质三级结构;通过确定一些保守区域的氨基酸 ,预测蛋白质的功能。
[结果 ]HpaGXoo蛋白由 139个氨基酸组成 ,分子量为 13 726.7 Da,理论等电点为 4.07。该蛋白分子式为 C569H896N174O212S5 ,酸性氨基酸残基总
数(Asp+Glu)为 10,碱性氨基酸残基总数(Arg+Lys)为 3;疏水性的总平均值(GRAVY)为-0.553。HpaGXoo二级结构中 , α-螺旋与无规则卷曲的数量与所占百分比分别为 34与 105、24.46%与 75.54%。可见 ,该蛋白富含无规则卷曲和α-螺旋结构。 HpaGXoo跨膜区域为零 , 且在跨
膜区域中的氨基酸序列期望值为 0.033 31,该结果在大于 18时才有跨膜区域 , 故该蛋白没有跨膜区域。 HpaGXoo蛋白没有信号肽存在。HpaGXoo蛋白存在于细胞质中的可能性为 21.4%,而在细菌壁膜间隙、外膜、内膜都无法确定是否有该蛋白的存在。HpaGXoo蛋白的二级结构
中主要为α-螺旋和无规则卷曲 ,并且占相当比例 ,而无 β -折叠存在。 HpaGXoo蛋白分子量小 ,只有 139个氨基酸残基 ,属于结构简单的蛋白 ,故推测其全蛋白可能就是一个功能结构域 ,或是一部分功能结构域。
[结论 ]该研究为深入研究HpaGXoo的分子结构及其结构与功能的关系奠定了基础。关键词 HpaGXoo蛋白;生物信息;结构分析;功能预测
基金项目 廊坊师范学院科学研究资助项目(LSZB200803)。
作者简介 任秀艳 (1977-),女 ,吉林九台人 ,博士 ,讲师,从事生物化学及分子植物病理学研究。 ＊通讯作者。
收稿日期 2009-12-21 修回日期 2010-02-20
75YANGJian-yuetal.AFLPAnalysisofPhylogeneticRelationshipofSalviaspp.inBeijing