全 文 ：nrDNA ITS 和 cpDNA rpl16 基因测序法
对六种鼠尾草的检测?
郭会芳1 , 阚显照2 , 张 韧1 , 3 , 陈鸿珊1
??
(1 中国医学科学院 北京协和医学院 医药生物技术研究所 , 北京 100050;
2 中国科学院植物研究所系统与进化植物学国家重点实验室 , 北京 100093 ;
3 卧龙岗大学生命科学院 , 澳大利亚 新南威尔士 卧龙岗 2522)
摘要 : 为从鼠尾草属植物中鉴别丹参品种 , 采用基因测序方法 , 用核糖体核酸内转录间隔区基因 ( nrDNA
ITS) , 编码核蛋白体大亚基多肽 L16 的基因 ( rpl16 ) 及叶绿体 DNA 上包含 trnL 以及 trnL 和 trnF 间隔区的
区域基因 ( trnL- trnF) 的序列 , 检测六种鼠尾草属新鲜植物。由于 nrDNA ITS和 rpl16 突变率较高 , 可以做
为 6 种鼠尾草的基源鉴定标记 , 依此设计了两对特异引物 , 从 6 种鼠尾草中鉴定出丹参 ( Salvia miltiorrhi-
za) 和云南鼠尾草 ( S. yunnanensis)。但 trnL- trnF突变率太低 , 未能用于鉴别。商品干燥中药材因加工和储
藏的方式致使 DNA 降解严重 , 基因测序法难于应用。
关键词 : 鼠尾草属 ; ITS基因 ; rpl16 基因 ; trnL- trnF 基因
中图分类号 : Q 945 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 03 - 345 - 06
Identification of Salvia Species by nrDNA ITS
and cpDNA rpl16 Sequence Analyses
GUO Hui-Fang1 , KAN Xian-Zhao2 , ZHANG Ren1 , 3 , CHEN Hong-Shan1 * *
( 1 Instituteof Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union of Medical College,
Beijing 100050 , China; 2 State Key Laboratory of Systematic and Evolutionary Botany,
Instituteof Botany, ChineseAcademy of Sciences, Beijing 100093 , China;
3 School of Biological Sciences, University of Wollongong, NSW 2522 , Australia)
Abstract: Three DNA regions were sequenced for testing six fresh plant samples of Salvia species . These three DNA re-
gionswere nrDNA ITS ( nuclear ribosomal DNA internal transcribed spacer) , chloroplast rpl16 ( thegeneencoding riboso-
mal protein L16 ) , and trnL- trnF ( the cpDNA region comprising the trnL and the intergenic spacer between trnL and
trnF ) . The results showed that the nrDNA ITS and rpl16 genes could provide novel informationfor origin identification of
Salvia species . Due to their higher mutation rates of these 2 gene markers, Salvia species-specific primerswere designed
and S. miltiorrhiza and S. yunnanensiswere identified . The trnL- trnF geneexpressed lowmutation rate, it couldnot iden-
tify the species . Since the damage of DNA by the pretreatments of the dry roots of Chinese herbs, it is hard to apply the
molecular markers to commercial samples for identification .
Key words: Salvia; ITS; rpl16; trnL- trnF
The dried root and rhizome of Salvia miltiorrhiza
Bunge (Danshen) has been used for hundreds of years
as a traditional Chinesemedicine ( TCM) , (Pharmaco-
poeia Commission of PRC, 2005 ) . Danshen is used
云 南 植 物 研 究 2008 , 30 (3) : 345～350
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07224
?
?? ?Author for correspondence; Tel : + 86 - 10 - 63010984 ; Fax: 86 - 10 - 63017302; E-mail : chenhs—10 @163 . com
Received date: 2007 - 09 - 29 , Accepted date: 2007 - 12 - 05
作者简介 : 郭会芳 (1983 - ) 女 , 在读博士研究生 , 主要从事抗病毒药物研究。Tel : + 86 - 1381 156 7956; E-mail : gracenlion@163 . com ?
Foundation item: Pharmaceutical Innovation and Traditional Chinese Medicine Modernization Program of China ( 2004AA2Z3342)
mostly for its cardioprotective and anti-atherosclerotic ef-
fects, but its componentshavebeen found to possessdi-
verse potential medicinal activities, including the inhi-
bition of HIV-1 integrase (Abd-Elazem et al. , 2002 ) .
Although only S. miltiorrhiza was recorded in the Chi-
nese Pharmacopoeia, thedried root and rhizomeof other
Salvia species such as S. yunnanensis C . H . Wright,
S. przewalskii Maxim ., S. trijuga Diels, S. digi-
taloides Diels, S. flava Forrest ex Diels and other spe-
cies have also been used and called Danshen . All these
wild Salvia are normally not cultivated in large scales
by GAP ( Good Agriculture Practice) and sometimes
mixed-up with Salvia miltiorrhiza due to the similar
morphology and similar efficacy . Extracts from different
species had different potencies for developingdrugs . In
theprogress of TCM modernization and for further de-
velopment of thisvaluable herbmedicine, it isof criti-
cal importance to accurately record the source of plant
materials and standardize the herb species and the me-
dicinal effects, so that the qualities of herb products
will bewell controlled . Sincemolecular identification is
under investigation anddevelopment recently, many mo-
lecular markers, especially sequences of fast-evolving
DNA regions, have been developed for plant taxonomic
studies at the species level (Shaw et al. , 2005) . We
made a preliminary test to evaluate the feasibility of this
method, using the nrDNA ITS region, chloroplast rpl16
gene and trnL- trnF region of the cpDNA as molecular
markers to identify the six Salvia species which are
used as Danshen in Yunnan provinceof China .
1 Materials and Methods
1 .1 Plant materials
All fresh Salvia samples aslisted inTable1 , werecollected
in the summer of 2005 in Yunnan Province . The fresh samples
were dried with silicagel in12 hours and kept coldfor longtime
storage . Voucher specimens have been deposited in the Herbari-
um of the Institute of Botany, Chinese Academy of Sciences,
Beijing (PE) . Commercial samples of S. miltiorrhizaBunge were
bought fromTongrentang drug store of Beijing .
1 . 2 ?DNA extraction, PCR amplification and sequencing
Total DNA was extracted from silica gel dried leaves using
theCTAB method following the protocol of Rogers and Bendich
( Rogers and Bendich, 1988) and used astemplate inpolymerase
chain reaction (PCR) . Similar total DNA extraction method was
applied to commercial samples except that PVP ( polyvinylpyrro-
lidone) was added to improve the quality of DNA . The nrDNA
ITS regionwas amplified with primers ITS1N (5′-GTC GTA ACA
AGG TTT CCG TAG G) modified from White et al. (1990) and
ITS4 ( 5′-TCC TCC GCT TAT TGA TAT GC) , the rpl16 gene
was amplified with primers rpl16F ( 5′-CCC?T TTC ATT CTT
CCC CTA TGT TG) and rpl16R (5′-GCT ATG CTT AGT GTG
CGA CTC GTT G) (Small et al. , 1998) and the trnL- trnF re-
gion with 5′trnLUAAF (5′-CGA AAT CGG TAG ACG CTA CG)
and trnFGAA ( 5′-ATT TGA ACT GGT GAC ACG AG) (Taberlet
et al. , 1991) . The PCR amplification was carried out in a vol-
ume of 25μl , containing5 - 50 ng of DNA template, 6 . 25 pmol
of each primer, 0 . 2 mM of each dNTP, 0 . 75 units of ExTaq
DNA polymerase ( TaKaRa, Dalian, China) . Amplifications
were conducted in a Tgradient 96 U thermocycler ( Biometre,
G?ttingen, Germany) with PCR regimes as follows: for ITS, one
cycleof 4 min at 70℃ , 4 cycles of 40 sec at 94℃ , 20 sec at
55℃ and 2 min at 72℃ , followed by 30 cycles of 20 sec at
94℃ , 20 sec at 55℃ and 2 minat 72℃ with afinal step for 10
min at 72℃ ; for rpl16 and trnL- trnF , they were the same ex-
cept for the annealingtemperature at 50℃ instead of 55℃ . The
PCR productswerepurified usingthe DNA Fragment Quick Puri-
fication?Recover Kit (DingGuo, Beijing) . Sequencing reactions
were performed with the same primers listed above . After precip-
itation in 95% EtOH and 3M NaAc ( pH 5 .2) , the sequencing
products were separated on a MegaBACE 1000 automatic DNA
sequencer (AmershamBiosciences, Buckinghamshire, UK) . For
nrDNA ITS and rpl16 , three to six individual plants per species
were sequenced and only one individual plant was sequenced for
trnL- trnF , all with both forward and reverse primers .
Table 1 Plant materials and GenBank Accession Nos . of the sequences
Taxa Voucher Locality
GenBank Accession Nos .
ITS rpl16 ?
S ?. yunnanensis C . H . Wright NO . 201757 {Kunming, Yunnan, China EF014344 EF014350 M
S ?. miltiorrhiza Bunge NO . 2017260 Beijng, China EF014345 EF014351 M
S ?. przewalskii Maxim NO . 2017266 Lijiang, Yunnan, China EF014346 EF014352 M
NO . 2017272 ?Deqin, Yunnan, China EF053400 EF053401 M
S ?. trijuga Diels NO . 2017254 Lijiang, Yunnan, China EF014347 EF014353 M
S ?. digitaloides Diels NO . 2017269 Lijiang, Yunnan, China EF014348 EF014354 M
S ?. flava Forrest ex Diels NO . 2017262 Lijiang, Yunnan, China EF014349 EF014355 M
643 云 南 植 物 研 究 30 卷
1 .3 Data analysis
Sequence alignments were made with Clustal X 1 .81
(Thompson et al. , 1997) and refined manually . Genetic distance
between Salvia species was estimated with Mega 3.1 (Kumar et
al. , 2004) based respectively on the ITS region and the rpl16
gene, using Kimura′s two-parameter model (Kimura, 1980) .
2 Results and Discussion
The quality of total DNA of commercial samples
was not good enough for amplifying . All PCR products
were amplified fromtotal DNA of fresh samples .Weob-
tained about 630 bp for the ITS region, 800 bp for rpl16
and 775 bp for trnL- trnF . Sequence alignments of the
former two DNA regions are shown in Fig . 1 and Fig .
2, respectively . The ITS region consists of the 5.8S
coding region and two internal transcribedspacers ( ITS1
and ITS2) .GenBank accession numbers assigned for the
sequences we determined are listed in Table 1 .
Among the six Salvia species, sequences of the
trnL- trnF region were nearly identical while those of
the ITS region and the rpl16 gene were more variable
(Fig . 1 , 2 ) . No intraspecific variation was detected,
although somespecies, like S. przewalskii , were repre-
sented by several individuals collected fromdifferent lo-
cations . The interspecific sequence divergence was
higher in the ITS region (0 . 005 - 0.065 ) than in the
rpl16 gene (0 . 004 - 0 .023) ( Tables 2 , 3 ) , and each
Salvia species was found to have auniquesequence in
the two DNA regions .This indicates that nucleotidese-
quences of DNA regions like nrDNA ITS and rpl16
could provide novel information for origin identification
of Salvia species due to their higher mutation rate .
Since the trnL- trnF region did not show enough varia-
tion, it might discount its useas amolecular marker for
authentication of Salvia species in general .
Fig . 1 Sequence comparison of the nrDNA ITS region among the six Salvia species . Dot indicates identical nucleotide or gap as in the
uppermost sequence . Hypen indicates an introduced gap . Arrow indicates theposition of species-specific primer .
Abbreviations: SD: S. digitaloides; SF: S. flava; SM: S. miltiorrhiza ; SP: S. przewalskii ; ST : S. trijuga; SY : S. yunnanensis
7433 期 GUO Hui-Fang et al .: Identification of Salvia Species by nrDNA ITS and cpDNA rpl16 Sequence . . .
Fig . 2 Sequence comparison of the rpl16 gene among the six Salvia species . Dot indicates identical nucleotideor gap as in the
uppermost sequence . Hypen indicates an introduced gap . Arrow indicates theposition of species-specific primer .
Abbreviations: SD: S. digitaloides; SF: S. flava; SM: S. miltiorrhiza ; SP: S. przewalskii ; ST : S. trijuga; SY : S. yunnanensis
Table 2 Genetic distance between six Salvia species
according to the ITS region sequences
1 ?2 3 4 ?5 16
1 ?. S 8. yunnanensis
2 ?. S 8. miltiorrhiza 0 k. 029
3 ?. S 8. przewalskii 0 k. 053 0 .051
4 ?. S 8. trijuga 0 k. 065 0 .060 0 . 046
5 ?. S 8. digitaloides 0 k. 055 0 .053 0 . 008 0 .041
6 ?. S 8. flava 0 k. 050 0 .048 0 . 006 0 .040 0 .005
To discriminate Salvia miltiorrhiza from S. y-
unnanensis and other related species in Yunnan fastly
and efficiently, species-specific primers were further
designed based on sequences of the ITS region and the
rpl16 gene . We found that the primer pair TYF ( 5′-
CGA CGC ACG TCC TCG GGC AGT) and TYR (5′-
Table 3 Genetic distance between six Salvia species
according to the rpl16 gene sequences
1 i2 3 4 5 6
1 . S. yunnanensis
2 . S. miltiorrhiza 0 ?. 005
3 . S. przewalskii 0 ?. 017 0 >. 015
4 . S. trijuga 0 ?. 023 0 >. 020 0 ]. 019
5 . S. digitaloides 0 ?. 015 0 >. 015 0 ]. 008 0 |. 019
6 . S. flava 0 ?. 016 0 >. 013 0 ]. 007 0 |. 017 0 .004
GGG CGC AAC ACG CGC AAT) (Fig . 1) specifically
amplified the ITS region of S. yunnanensis at the an-
nealing temperatureof 55℃ ( Fig . 3 : A) , while prim-
ers RYF-1 (5′-TCA AAC AAA ATA GAT GAA T) and
RYR ( 5′-AGT TAA ATC AGT CAT ATC ) ( Fig . 2)
specifically amplified the rpl16 gene of S. miltiorrhiza
843 云 南 植 物 研 究 30 卷
Fig . 3 Specific amplification of parts of thenrDNA ITS region ( A ) and the rpl16 gene (B) . Separation of the fragments was achieved by
electrophoresis on 1 .5 % agarosegels, and bands were visualized with GoldViewTM ( SBS Genetech, Beijing) . M : 1 . 5 kb DNA marker .
Abbreviations: SD: S. digitaloides; SF: S. flava; SM: S. miltiorrhiza ; SP: S. przewalskii ; ST : S. trijuga; SY : S. yunnanensis
at the annealing temperature of 40℃ ( Fig . 3 : B ) .
Thus, primers TYF and TYR differentiated S. y-
unnanensis fromtheother five Salvia species and prim-
ers RYF-1 and RYR offered an alternative method for
the Chinese Pharmacopoeia to authenticate S. mil-
tiorrhiza .
Oneof the problems that restrain the development
of traditional Chinese medicine is the authentication of
rawmaterial in the field and on the commercial mar-
kets . Ways basedon DNA sequencing may offer an al-
ternative andmore reliablemethod for the identification
of Chinese medicinal materials ( Zhao et al. , 2002;
Cao and Komatsu, 2003; Chen et al. , 2002; Wang et
al. , 2005; Lau et al. , 2001; Sun et al. , 2004; Ding
et al. , 2002; Kojoma et al. , 2002; Hosokawa et al. ,
2004; Xu et al. , 2006 ) and this will lead to another
set of standardization of traditional Chinese medicine .
However, DNA-based polymorphism may not handle
the identification all by itself . Morphological and
chemical studies are indispensable since most Chinese
medicinal materials on the market have been pro-
cessed . The condition of storage and processing of raw
materialsoften result in DNA degradation . All thesam-
ples tested were live plants collected in the field and
dried by silica gel soon after . Samples bought fromthe
market had also been tested in our laboratory, but un-
fortunately, thequality of DNA was not qualified to be
amplified . With the fact of DNA degradation, species-
specific primers aimed for shorter DNA fragments
should be designed, which may give satisfactory re-
sults .
In conclusion, we have been able to molecularly
distinguish some Salvia species used as TCM plants
with two markers . However, since it′s hard to apply
themarkers to the commercial samples, maybemolecu-
lar identification based on sequence analyses couldn′t
shoulder the task of identifying Chinese medicinal ma-
terials all alone . DNA-based polymorphism may offer
an efficient choice for authenticating medicinal herbal
species for researchers, but further investigation is still
in need for developing its market value .
Acknowledgements: We thank Ms . J-X Li and Drs . J Zhao,
Y-Z Cun, S-S Wang and Y-F Li for their help with sample col-
lection . Dr . H Gao is thankedfor her assistance in the laboratory
work . Especiallywethank Prof . Xiao-Quan Wangof IB-CAS for
his useful guidance and discussion .
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