全 文 ：一种从苏铁叶片中有效提取 RNA 的方法
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李 璐1 , 付乾堂1 ,2 , 余迪求1
??
(1 中国科学院西双版纳热带植物园 , 云南 昆明 650223 ; 2 中国科学院研究生院 , 北京 100049)
摘要 : 由于苏铁 ( Cycasrevoluta) 叶片中含有大量的多糖多酚等次生代谢物 , 常规 RNA 提取方法很难获得
优质的 RNA。在常规的 CTAB 法中加入了硼砂和β- 巯基乙醇来消除多酚和多糖的干扰 , 得到了一个从苏
铁叶片中有效提取 RNA 的方法 , 每克鲜叶片可获得约 930μg RNA。A260?280 和 A260?230 的纳米波长的吸收比值
都约为 2 , 表明 RNA 的质量较好。获得的 RNA 可用于 Northern blot和反转录 PCR 等分析 , 也说明 RNA 的质
量比较好。此外 , 改进的提取方法也适合于含有次生代谢产物的其它植物 , 同样可以获得优质 RNA。
关键词 : 苏铁 ; 硼砂 ; RNA 提取
中图分类号 : Q 943 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 05 - 593 - 04
An Effective Protocol for the Isolation of
RNA from Cycad Leaves*
LI Lu1 , FU Qian-Tang1 , 2 , YU Di-Qiu1 * *
( 1 Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223 , China;
2 GraduateUniversity of Chinese Academy of Sciences, Beijing 100049 , China)
Abstract : Conventional RNA extractionmethods havebeen shown toproduce poor-qualityRNA whenappliedto Cycasrev-
oluta becauseof abundant secondary metabolites included . Withmodification of the standard cetyltrimethylammonium bro-
mide ( CTAB) method by addingborax (disodiumtetraboratedecahydrate) andβ-mercaptoethanol to eliminatethe interfer-
enceof polyphenol and polysaccharides, an effective protocol was developed . This modified protocol could extract high
quantities of RNA from cycad leaves at about 930μg per gram of fresh weight . Both A260?230 and A260?280 ratioswerearound
2 , indicatingthat RNA is of high quality . The RNA quality was confirmed by Northern blotting analysis and reverse tran-
scription polymerase chain reaction ( RT-PCR) . The modified protocol could be successfully extended to other plants con-
taining secondary metabolites .
Key words: Cycasrevoluta; Borax; RNA extraction
Extraction of high-quality RNA is necessary for
making cDNA library, isolating genes by RT-PCR , or
investigatinggene expression profile . When performing
the project“ construction of the cDNA library for the
valuable plants in Yunnan”, wefound it difficult to ex-
tract RNA from the leaves of Cycas revoluta ( Cyca-
daceae) using availableconventional protocols . The ex-
tract was highly viscous and brown becauseof the large
amounts of polysaccharides, polyphenols and other uni-
dentified compounds . Phenolic compounds are readily
oxidized to form covalently linked quinines ( Loomis,
1974) , and avidly bind nucleic acids . The same situ-
云 南 植 物 研 究 2008 , 30 (5) : 593～596
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07272
?
?? ?Author for correspondence . E-mail : ydq@ xtbg. ac. cn; Fax: + 86 - 871 - 5160916; ph: + 86 - 871 - 5178133
Received date: 2007 - 11 - 21 , Accepted date: 2008 - 03 - 13
作者简介 : 李璐 ( 1974 - ) 女 , 博士 , 主要从事功能基因的起源与进化。 ?
Foun ?dation items: The Programs of the Natural Science Foundation of Yunnan Province (Grant No . 2005C0034Q) , Demonstration of Collecting&
Conserving Standardization and Sharing of Important Wild Plant Germplasm Resources ( Grant No . 2004DKA30430 , Grant No .
2005DKA21006) , and the Knowledge Innovation Program of Chinese Academy of Sciences
ation was also found in other two woody gymnosperms
such as Ginkgo biloba ( Ginkgoaceae) , Taxus waclli-
chiana ( Taxaceae) and three angiosperm species, in-
cluding theornamental orchid Cypripediumflavum (Or-
chidaceae) and two horticultural plants endemic to
Yunnan: Mussella lasiocarpa (Musaceae) , and Lucu-
lia gratissima ( Rubiaceae) . In the present study, we
successfully isolated high-quality and high-quantity
RNA fromthesesix plants with amodified CTAB meth-
od . Northern blotting and RT-PCR analysis were used
to confirmthe quality of the extractedRNA from C . re-
voluta .
1 Materials and methods
1 .1 Materials
Young and mature leaves from six plants ( C. revoluta,
G. biloba, T. wallichiana, Cy. flavum, L. gratissima, M. las-
iocarpa) were collected in Kumming Botanical Garden of Kun-
ming Institute of Botany, the Chinese Academy of Sciences .
Some cycad leaves harvested were treated with salicylic acid
(SA) for 12 hr for Northern blotting analysis . All samples were
frozen immediately with liquid nitrogen after collection and then
stored at - 80℃ until use .
1 . 2 RNA isolation
Modified extraction buffer: 2% (w?v) CTAB , 0 .125 M di-
sodium tetraborate decahydrate, 0 . 1 M Tris?HCl ( pH 7 .5 ) , 2 M
NaCl, 0 . 025 M EDTA , and2% ( v?v)β-mercaptoethanol (add-
ed just before use) ; 4 M LiCl; 5 M NaCl ; 70 % (v?v) ethanol .
These solutions were treated with 0.1 % ( v?v) DEPC . Phenol?
Chloroform?isoamyl alcohol (25∶24∶1 [ v?v] ) , 0 .1 % DEPC-
treated water .
Modified protocol: Three grams of frozen leavesweregrind-
edto a fine powder and added to 10 mL pre-warmed extraction
buffer, shaken vigorously and incubated at 65℃ for 6 min .
When the homogenate was cooled for 10 min on ice, an equal
volume of phenol?chloroform?isoamyl alcohol ( 25∶24∶1 ) was
added andmixed gently . Following centrifugation at 10 000 g for
20 min at 4℃ , the collected upper phase was precipitated with
an equal volumeof 4 M LiCl at 4℃ for 4 hours . After centrifuga-
tion at 10 000 gfor 20 min at 4℃ , the precipitate wassuspended
in 10 ml DEPC-treated water, and precipitated with 2 .5 volume
of ethanol and 1?10 volume of 5 M NaCl at - 80℃ for 1 hour .
After the same centrifugation as last time the pellet was washed
with 70% ( v?v) ethanol and air-dried on ice for 10 min . And
then, the pellet was resuspended in an adequate volume of dou-
ble-distilled DEPC-treated water . The RNA is ready for use or
can be stored at - 80℃ for up to1 month .
1 . 3 RNA analysis
The purified total RNA was quantified with a spectropho-
tometer atwavelengths of 230 nm, 260 nm, and 280 nm . The in-
tegrityof total RNA was verified by running sample on a 1 .5%
formaldehyde?agarose gel .
1 . 4 Northern blotting analysis
The Northern blottingprotocol was based on themethod de-
scribed by Sambrook et al. ( 1989 ) . Partial CrWRKY sequence
was obtained byPCR usingthedegenerateprimersWRKY1-FP &
WRKY 2-RP ( Borrone et al. , 2004) from the genomic DNA of
C. revoluta and cloned into pUCm-T Vector (Sangon, Shang-
hai ) . The positivecloneswereconfirmed by sequencing and des-
ignated as CrWRKY1 , CrWRKY2 and CrWRKY3 , respectively .
The partial sequences of the three CrWRKY genes were used as
probes in Northern blottinganalysis respectively .
1 . 5 RT-PCR analysis
The single-stranded cDNA product was synthesized fromthe
DNAase-treated RNA of C. revoluta usingthe AMV reversetran-
scriptaseaccording to themanufacturer′s instructions (Fermentas
Corp .) . The product was used to amplify the CrrbcL gene with
the primers derived from the coding region of CrrbcL gene
( CrrbcL-F: 5′-AGCAGCGTTCCGAGTAACTCCT-3′and CrrbcL-
R: 5′-TGAGCCAAGCTGGTATTTGCA-3′) . The amplified prod-
ucts were separated on a 1 .5 % agarose gel and visualized by
ethidium bromide staining .
2 Results and discussion
Using themodified protocol , pureRNA was easily
acquired within 8 hours . It efficiently eliminated most
of the interference and produced white and water-solu-
ble RNA precipitates with high-yield ( approximately
0 .42 - 0 .956 mg?g fresh wt) ( Table 1 ) . Both A260?230
and A260?280 ratios were around 2 indicating that the
RNA preparations were free of polysaccharide?polyphe-
nol and protein contamination, respectively (Table 1) .
A formaldehyde-denatured agarose gel was used to
check the integrity of ribosomal bands visulalized in
Table 1 Quality and yield of total RNA isolated from six plants*
Plant species A260 ??280 A260?230 μg RNA?g FW
C ?. revoluta 1 . 90±0 . 023 1 ?. 88±0 .026 956±35 \. 6
G ?. biloba 1 . 99±0 . 019 2 ?. 27±0 .021 921±26 \. 9
T ?. wallichiana 1 . 85±0 . 018 2 ?. 30±0 .014 570±19 \. 8
M ?. lasiocarpa 2 . 08±0 . 021 2 ?. 11±0 .068 420±23 \. 5
L ?. gratissima 2 . 03±0 . 032 2 ?. 26±0 .042 640±31 \. 2
C ?. flavum 1 . 97±0 . 041 2 ?. 04±0 .031 675±21 \. 9
* Values are means±SD ( n= 6) .
495 云 南 植 物 研 究 30 卷
Fig . 1 Electrophoretic analysis of RNA isolated from six plants
C : C . revoluta; G: G. bioloba; T: T. wallichiana;
M: M . lasiocarpa; L : L . gratissima;
Cy: Cypripediumflavum .
Fig . 2 Electrophoretic analsis of RNA from 4 plants
by two different methods
C1 , G1 , T1 , and Cy1 : by standard CTAB method ( Chang et al. ,
1993 ) ; C2 , T2 , G2 , and Cy2 : by TRizol Kit ( Invitrogen)
Fig . 1 . The two main distinct bands ( 28S rRNA &
18S rRNA) could bevisualizedon thegel without obvi-
ous degradation .
In this study, we used theTriZol kit ( Invitrogen)
and conventional CTAB method ( Chang et al. , 1993)
to extract RNA from these six plants respectively .
However, these two methods gave poor results and
failed to yield useable RNA for further investigation
(Fig . 2 ) . The pellets were highly viscous and brown
in gymnosperm plants and orchid . When the improved
CTAB protocol applied, high-quality and high-quantity
RNA was extracted fromthese plants . The most useful
modification is the addingof borax andβ-mercaptoetha-
nol into the standard CTAB extraction buffer . In order
to eliminate the oxidation of polyphenol , we tried dif-
ferent reagents, eg: PVP ( polyvinylpyrrolidone) , DTT
( dithiothreitol ) , β-mercaptoethanol , and borax . Our
results showed that the borax, together withβ-mercap-
toethanol , was more efficient than theother reagents to
eliminate the interference of brown complexity . Previ-
ous studies in cotton have confirmed the significance of
borax in preventingsample fromoxidation, together with
other reagents, such as Nonident-40 ( NP-40 ) , PVP,
proteinaseK , and DTT ( Wan and Wilkins, 1994; Wu
et al. , 2002) . Therefore, usingborax andβ-mercapto-
ethanol in the CTAB extraction buffer is an effective
modification to gain clean RNA fromthe six plants .
In addition, it is an efficient protocol to obtain
pure RNA . The RNA can be directly isolated from
DNA , proteins, and other secondary productions after
LiCl precipitation . This protocol will be finished within
one day . And then the RNA could be washed by 70%
ethanol and resuspended in DEPC-treated water for
Northern blotting . If RNA is used for cDNA library
construction or RT-PCR, it should be precipitated by
ethanol?NaCl and then washed . Long-term storage of
RNA canbeachieved by additionof 2 .5 vol of absolute
ethanol and 1?10 vol of 5 M NaCl .
The RNA isolated from cycad leaves was used in
gene expression analysis by Northern blotting . WRKY
genes encode a superfamily of plant-specific transcrip-
tional factors, regulating physiological responses to bi-
otic and abiotic stresses ( Hara et al. , 2000; Rushton
and Somssich, 1998) and senescence as well as devel-
opment ( Camerom, 2002; LagacéandMatton, 2004) .
SomeWRKY genes expressions wereinducedby molec-
ular signal SA to be associated with plant defense re-
sponses (Dong et al. , 2002) . Studies on WRKY genes
reported are restricted to only a few plant species such
as Arabidopsis, rice, and tabacco (Yu et al. , 2001;
Hara et al. , 2000; Zhang et al. , 2004 ) . For this rea-
son, we took an approach of analyzing the expression
profiles of the three WRKY gene fragments
( CrWRKY1 , CrWRKY2 , CrWRKY3) in bothuntreated
and SA-treated leaves of cycad . Thegood-quality RNA
of SA-treated or untreated leaves from cycad was used
in WRKY genes expression analysis by Northern blot-
ting, respectively (Fig . 2) . The analysis revealed that
the activities of the three WRKY gene fragments were
not associated with SA , since the expression levels of
5955 期 LI Lu et al. : An Effective Protocol for the Isolation of RNA from Cycad Leaves
the three genes didn′t show any significant difference
between in SA-treated and untreated leaves . The re-
sults implied that the threegenes might not be involved
in SA-mediated disease resistant pathway .
The good quality of the RNA was also proved by
confirmation fromRT-PCR in cycad (Fig . 3) . By use
of the first strand cDNA , a part of the CrrbcL gene
(around 400 bp) was successfully cloned .
Fig . 3 Northern blotting analysis of RNA extracted from cycad leaves
0: RNA from untreated leaves; SA : RNA from SA-treated leaves
Fig . 4 Agarose electrophoresis of the RT-PCR products
1 . rubulose 1 , 5-bisphosphate carboxylase large subunit gene
rbcL (400 bp) , M . 100-bp DNA ladder .
In conclusion, the modified protocol could yield
pure RNA from both mature and young leaves of these
six plants .
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