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An Optimized Chloroplast Isolation and Chloroplast DNA Extraction Protocol for Chinese Wild Grapes

中国野生葡萄叶绿体分离及叶绿体DNA提取的研究



全 文 :书!"#$%&
,2016,36(7):1464-1469
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kz.

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RŸ‚

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4J=K$
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犃狀犗狆狋犻犿犻狕犲犱犆犺犾狅狉狅狆犾犪狊狋犐狊狅犾犪狋犻狅狀犪狀犱犆犺犾狅狉狅狆犾犪狊狋犇犖犃
犈狓狋狉犪犮狋犻狅狀犘狉狅狋狅犮狅犾犳狅狉犆犺犻狀犲狊犲犠犻犾犱犌狉犪狆犲狊
XIEHaikun,JIAOJian,FANXiucai,ZHANGYing,JIANGJianfu,
SUNHaisheng,LIUChonghuai
(ZhengzhouFruitResearchInstitute,ChineseAcademyofAgriculturalSciences,Zhengzhou450009,China)
犃犫狊狋狉犪犮狋:Matureleavescolectedfrom犞犻狋犻狊犱犪狏犻犱犻犻,犞.犪犿狌狉犲狀狊犻狊,犞.犺犲狔狀犲犪狀犪and犞.犮犺狌狀犵犪狀犲狀狊犻狊
wereusedforchloroplastisolationandcpDNAextractioninthisstudy.Thetwomethodswerethecolumn
plantchloroplastDNAoutandmodifiedhighsaltlowpHmethod,andtheresultswerecomparedwitheach
other.(1)BothmethodshadseparatedthechloroplastofChinesewildgrapes,butthemodifiedhighsalt
lowpHmethodobtainedhigherconcentrationandlessimpurityofchloroplastthanthatofcolumnplant
chloroplastDNAout.SothemodifiedhighsaltlowpHmethodwasmoresuitableforchloroplastisolation.
(2)ThevalueofOD260/OD280ofcpDNAextractedbythecolumnplantchloroplastDNAoutwasbetween
1.28and1.36,andtheconcentrationwasbetween4.2ng·μL
-1and7.8ng·μL
-1,whichdidnotmeet
thedemandofsubsequentchloroplastgenomesequencing.Incontrast,thevalueofOD260/OD280ofcpDNA
extractedbythemodifiedhighsaltlowpHmethodwasbetween1.84and1.90andtheconcentrationwas
between2514.4ng·μL
-1and4133.7ng·μL
-1,sothecpDNAextractedinthiswaywasextremely
highqualityandpure.Asaresult,thecpDNAextractedbythemodifiedhighsaltlowpH methodmeet
thedemandofsubsequentchloroplastgenomesequencing.Asaconclusion,themodifiedhighsaltlowpH
methodisolatedintactchloroplastandextracthighqualitycpDNAofChinesewildgrapessimplyand
quickly.AndthecpDNAmeetthedemandofsubsequentchloroplastgenomesequencing.Itwasalsoa
criticalsteptomakefurtherresearchofchloroplastgenomesof犞犻狋犻狊L.
犓犲狔狑狅狉犱狊:Chinesewildgrapes;chloroplastisolation;cpDNA extraction;columnplantchloroplast
DNAout;modifiedhighsaltlowpHmethod
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10mmol/LÏÐÑÎÒ,0.0125 mol/L Ó Ô,
50mmol/L TrisHCl (pH 8.0),7 mmol/L
Na2EDTA(pH8.0),1% PVPK40ÕÖ×ØÙÚ
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(狑/狏),0.1%BSA ÕÝÍÞßà (狑/狏),
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…(20min,ˆ7Þ;Çé…(1ê。
(4)Á‡:4℃、3070g(þÿ!63500g),…
(20min,û7Þ。
(5)«ø:ëø\]6ʾ42mLº»oBuff
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20min,û7Þ。öèkz.\]6ʾ8mL
BufferC[100mmol/LNaCl,100mmol/LTrisHCl
(pH8.0),50mmol/LNa2EDTA,1mmol/LDTT
(DTTÆÊ)]、1.5mL20% SDS、20μLβ3
º×ã
、30μLßà4 K(10mg/mL),„5¾,
55℃#67689ä:,,kz.„12,;<
=cpDNA。
12Š>¤

r12ž—6 5 min,¤Ê¾
1.5mLKAc(pH5.7),?@5¾,ö—630min;
4℃、5260g(þÿ!610000g),…(15min,ÆA
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:\]cpDNA。
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20min,û7Þ,cpDNA。ÆNOPQr\]
R=

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×ã

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cpDNALž6ˆ2μL,GôÎßà\¦] (^ 7
Thermoª«,ND1000Y)7¦ÀôÎQŸ,Æ1×
TEBuffer(pH 8.0)X_àͯ,ó` OD260/
OD280、OD260/OD230†RŸ•–。
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iPQ

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2.2 456789犮狆犇犖犃[\]^
N 6 7 g K P Q cpDNA o OD260/OD280、
OD260/OD230 lRŸ•–²Ö(­1)¸ f|=,2?
ƒocpDNA}?wò~F‹。ƒI‡
ˆocpDNARŸ•–G4.2~7.8ng·μL
-1
ž

OD260/OD280ªG1.28~1.36ž,‚ì«ŠDNA
ovª1.8~2.0,©OD260/OD230ªG0.44~0.67
ž

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2.07~2.14ž,àì€c$o‚ª2.0,°®Õ
6641 ! " # $ % &                   36‰
x1 xy#$kz.DNAout(Ⅰ)l~€‚pHƒ(Ⅱ)„…d?gKPQkz.‹,xr
Fig.1 Chloroplastmicrographsof4grapesspeciesseperatedbycolumnplantchloroplastDNAout(Ⅰ)
andmodifiedhighsaltlowpHmethod(Ⅱ)
%

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a1 2bTU@AB犮狆犇犖犃cd=efg
Table1 UVscanningresultsofcpDNAextractedbytwomethods
?U
Species
‡ˆƒ
Method OD260
/OD280 OD260/OD230
RŸ•–
Concentration/(ng·μL-1)
hPQ
犞.犱犪狏犻犱犻犻
Ⅰ 1.30 0.58 7.8
Ⅱ 1.84 2.10 3708.5
iPQ
犞.犪犿狌狉犲狀狊犻狊
Ⅰ 1.35 0.67 5.6
Ⅱ 1.90 2.14 2876.0
jkPQ
犞.犺犲狔狀犲犪狀犪
Ⅰ 1.36 0.51 4.2
Ⅱ 1.87 2.07 2514.4
mnPQ
犞.犮犺狌狀犵犪狀犲狀狊犻狊
Ⅰ 1.28 0.44 4.8
Ⅱ 1.89 2.13 4133.7
˜
:Ⅰ、Ⅱ„wÍ5xy#$kz.DNAoutl~o€‚pHƒ
Note:ⅠandⅡstandforthecolumnplantchloroplastDNAoutandthemodifiedhighsaltlowpHmethod
x2 xy#$kz.DNAout(Ⅰ)l€‚pHƒ
(Ⅱ)‡ˆd?PQcpDNAklx™
Fig.2 ElectrophoresispatternofcpDNAof4grapes
extractedbycolumnplantchloroplastDNAout(Ⅰ)and
modifiedhighsaltlowpHmethod(Ⅱ)
  B3š›7œÌP̈́…PQkz.†‡ˆ
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±²³67gKPQ´µkz.†€RŸkz.
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iPQ

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.

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ƒ„…=67gKPQokz.†‡ˆocpDNA
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okz.†‡ˆocpDNA,œ OD260/OD280ªG
1.84~1.90ž,RŸ•–s2514.4~4133.7ng
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nomes[J].犃狀狀狌犪犾犚犲狏犻犲狑 狅犳 犌犲狀犲狋犻犮狊,1985,19(1):
325354.
[2] ² ³,´ åµ,¶ ·,ù.9ægK67¸¹º.kz.
DNA狋狉狀犙狉狆狊16§êKF†œÐъð„ñ[J].ÐÑ,
2012,34(11):14751483.
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8641 ! " # $ % &                   36‰
犎犲狉犲犱犻狋犪狊,2012,34(11):14751483.
[3] TOMOTARON,VAUGHANDA,KOHLCHIK.Phyloge
neticanalysisof犗狉狔狕犪species,basedonsimplesequencere
peatsandtheirflankingnucleotidesequencesfromthemito
chondrialandchloroplastgenomes[J].犜犺犲狅狉犲狋犻犮犪犾犪狀犱犃狆
狆犾犻犲犱犌犲狀犲狋犻犮狊,2005,110(4):696705.
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