Download DNA molecular identification

Development and application of DNA molecular
identification technology in TCM
Xu Hong
Institute of Chinese Material Medica
Shanghai University of Traditional Chinese Medicine
Contents for introduction
 Development of identification technology
in Traditional Chinese Medicine (TCM).
 Application of DNA molecular identification
technology in TCM.
 Studies on the Authentication of Caulis Dendrobii by
DNA Molecular Markers.
Induction

Importance: Chinese Traditional Medicine (TCM) has a more than
5,000 years history，due to various name and complex botanical origin
make them very difficult to differentiate. As a old research field,
species identification is still a growing field of interest in the study of
TCM.

Conventional identification methods:
Taxonomic identification
Authentication by Characters
Authentication by microscopic characters
Chemical analyses

New identification methods:
DNA molecular identification
Characteristic of markers
phenotype
subjective
• Shape、colour、odor—morphologic markers
• tissue、cell— cellular markers
• Chemical component and content—metabolic product markers
Effect of gene with environment together
polymorphic DNA — molecular markers
genotype
objective
DNA techniques for species identification
1. DNA sequencing
2. Random amplified polymorphic DNA (RAPD)
3. Inter-Simple Sequence Repeat (ISSR)
4. Restriction Fragment Length Polymorphism (RFLP)
5. Amplified Restriction Fragment Length Polymorphism (AFLP)
• PCR and automated DNA sequencing made
TCM identification at the DNA level possible..
DNA markers used for TCM identification:
• DNA sequencing marker：
Show the difference of aim gene directly ， information is
accurate and universal
1. chloroplast DNA (cpDNA)：evolves relatively slowly, moderate
to detect the genetic variation and relationship of high levels, such
as intra–family, family and upward family level. matK、atpB、
rps1b、rbcL、ndhD、trnK、trnF、trnT,
2. mitochondria DNA(mtDNA)：mainly applied in phylogenetic and
evolutionary studies of animal, as well as the identification of
animal medicines. cyt-b、12Sr DNA are most important markers.
3. nuclear DNA(nDNA)：important DNA markers for phylogeny
and identification of angiosperms. Further ,we will give emphasis to
rDNA.
Genetic structure of rDNA (ribosomal DNA) Repeat
IGS = intergenic spacer; includes the promoter region for RNA Pol I complex
ITS = internal transcribed spacers
5’
ETS
ITS1
ITS2
26S
18S
3’
IGS
18S
5.8S
In angiosperms, rDNA are organized in long tandem repeats, with
each containing a single transcribed region spanning the 18S, 5.8S,
and 26S rDNA, ITS1 and ITS2, and IGS. Although the 18S, 5.8S, and
26S rDNA are highly conserved, the ITS regions are variable in
different genus, species, even populations. Thus, the diversity of the
ITS region is widely used as a molecular marker for species
authentication and polygenetic analysis.
DNA fingerprint marker：
show the gene difference indirectly by DNA length through agarose
gel electrophoresis.
 RAPD marker
95%
different gene information from nuclear DNA, used for
indicating
the
genetic
variation
of
relative
individual,
but stability and reproducibility is not good.
 SCAR marker：clone sequencing of polymorphic RAPD product,
design primers to amplify the diagnostic RAPD region. stability and
reproducibility is more higher, so it is much more applied.
 RFLP
 AFLP
Application of DNA molecular marker on TCM identification
Plant medicine
DNA sequencing marker
RAPD marker：renshen，sanqi
rDNA ITS：Renshen，
xiyangshen，didancao, pugongyin，
dahuang ,xuelian,chongcao,
shanjiang，shihu, daqing, wuweizi,
and so on.
rDNA 18S: chuanxing，renshen，
banxia
mat K ：shihu，chuanxiong
trnK：baizhu,ezhu.
trnL-trnF: rougui
rbcL: banxia，tiannanxin
gancao，yinyanghuo, tiexianlian,
canzhu,congcao，huanglian，
sanmaidong, xixin, xihonghua，
tianhuafen, yujin, dangshen,
niubangzi, huangqi, huangqi n,
yangchunsha, hezi, gouqi, houpo,
xiangmao, shihu, jinxianlian, niuxi,
dihuang, shegan
SCAR marker：renshen, shihu
Animal medicine
Chinese Prescription
cyt-b, 12S rDNA:
Lurong ， Lubian ， Fushe,
Jinqianbaihuashe, Wushaoshe,
RAPD marker：
Shedan, Guiban,Guijia, Haima,
Yu-Pin-Feng-San
Jinneijin
Studies on the Authentication of Caulis Dendrobii
by DNA Molecular Markers
Induction
• Importance: Caulis Dendrobii(Shihu, stem of Dendrobium) is an
important traditional Chinese medicine that has commonly been
used as a tonic and diuretic in many Asian countries for centuries.
• Origin: originates from Dendrobium genus. the stems of D.
loddigesii, D. firmbriatum, D. chrysanthum, or D. nobile and related
species are listed as the origin of “Shihu” (Chinese Pharmacopoeia
2005).
Introduction about Dendrobium
 Dendrobium is one of the largest
families of orchids and creates much
diversity. They are found only in the
Eastern Hemisphere and range from
Australia, throughout the South
Pacific and Phillipines, Southeast Asia,
and India, and a small representation
in Japan.
长苏石斛
齿瓣石斛
金钗石斛

About 76 species and
varieties are identified in China,
and grow mainly in the north
from Qinling mountain.
鼓槌石斛
Introduction about Dendrobium
 Fresh and dried stem of more than 30
大苞鞘石斛
Dendrobium species carry the name
Shihu on the market of herbal medicine.
 Some of them are ornamentals
黄花石斛
翅萼石斛
翅梗石斛
肿节石斛
勐海石斛
Species recorded by different
edition of Chinese Pharmacopoeia
(from 1953 to 2005 )
Dendrobium nobile
D. officinale
D. loddigesii
D. fimbriatum
D. chrysanthum
Medicinal situation of Shihu
 Its main function is to benefit the stomach and promote the production
of body fluid, moisten the lung and relieve a cough, and resist cancer.
 “Shihu” is divided into two groups “Huangcao Shihu” and “Fengdou
Shihu” according to different processing methods. They originate
mainly from natural sources, and a large quantity is needed in China,
particularly in the south of China.
Huangcao
Fengdou
Problem
1.
The fresh stems of Dendrobium species, especially the processed
and dried stem have very similar morphological and anatomical
characteristics, and the traditional authentication of different
Shihu samples is therefore far from reliable.
2.
In addition, the chemical constituents of many Dendrobium
species are still unknown, as proper chemical analysis methods
have not been developed.
3.
However, the determination of the botanical origins of different
Shihu samples and their quality control through morphological
and chemical studies is fraught with difficultly.
Why use the DNA molecular markers?
 With the rapid development of biological techniques, many studies
indicated that DNA diversity might be used as a valuable source not
only for the evidence of biological phylogeny, but for identifying
crude medicine as well
 DNA-based methods depend on genotype rather than phenotype,
produce results that are not altered by the environment, and require
only a small amount of material. So, the DNA-based polymorphism
assay may offer an alternative method for the identification of herbal
medicine
What will we focus on?
 Which marker should we chose ? screened the speciesspecific markers for species identification of Shihu.
The ITS regions of Dendrobium species were sequenced
and compared to explore the possibility of using them
to differentiate these species.
 Further using this markers to the dried commercial
sample for identifying the source plant.
Part I:
Sequencing of ITS of Dendrobim
Aim: To evaluate whether rDNA ITS region could be
used as the molecular markers for Dendrobium
species identification
1. Extraction of total DNA
3.0 kb
1.5 kb
1. Dendrobium thyrsiflorum(Kit)
2. D. brymerianum(Kit)
3,4. D. nobile
(Lijiang, Yunnan；3. CTAB，4. Kit )
5. D. fimbriatum(Kit)
6,7. D. capillipes(6. CTAB，7. Kit)
Material: All materials were collected from different regions of
China, total 35 species
Brief summary: DNA was extracted from fresh leaf samples
using CTAB or Kit procedure. The quantity and quality of DNA
samples were checked on 1% agarose gel through comparison
with lambda DNA standards and by UV spectroscopy.
2. PCR amplification of ITS
Using primers constructed
from conserved regions 18S and
26S rRNA, a fragment of about
700bp
was
specifically
amplified, including the part
sequence of l8S and 26S rDNA,
the ITS1 and ITS2 regions, and
±700 bp
the 5.8S rDNA.
P26S
18S
ITS1
5.8S
ITS2
P18S
P1(P18S 3’): 5’-CGT,AAC,AAG,GTT,TCC,GTA,GGT,GAA,C-3’
P2(P26S 5’): 5’-TTA,TTG,ATA,TGC,TTA,AAC,TCA,GCG,GG-3
26S
• Sequencing: The PCR products were
purified using a Purification System and then
sequenced directly.
• Sequence analysis: The sequences were
aligned and compared using the Clustal W
programs and analyzed using the MEGA2
programs.
Result and discussion
1. Alignment of ITS1 and ITS2
ITS1
DSC
DCX
DDE
DTH
DH(YN)
DH(HN)
DA(GX)
DA(YN)
DAV(BN)
DAV(JL)
DBR
DFB
DCA
DCT
DFL
DN(YN)
DN(GX)
DN(HN)
DN(GZ)
DN(SC)
DEP
DCF
DWS
DEL
DAF
PHC
61
120
TGCTGCG-AC ATAATCCATC CAAGTCGTCG CCTCATCCCA TCTTCGGGGC GGGGA-CGCG
......AG.A .A...T.... .C.....GT. .......... A.C.T....T ........T.
.T.C.T...T GA.CG..... .C......TA ....--AT.C C...A...T. .A....T...
.T.C.T...T GA.CG..... .C......TA ....--AT.C C...A...T. .A....T...
....T....T .A.....G.. TC........ .........C ....T..... ....G.....
.........T .A........ TCT.....T. .........C ....T..T.. ....G.....
.......A.A .AG.....G. .C........ ....GC.... G.C.T..... .......C..
.......A.A .AG.....G. .C........ ....GC.... G.C.T..... .......C..
.......A.T .A........ .G.......T .......... .TC.TT.... ......T...
.......A.T .A........ .G...T...T .......... ..C.TT.... ......T...
.......A.T .A........ .C.A.TA... .......... ..C.A..... ........T.
..TC.T...T .A........ .C....A.T. .T.......C .TC.--.... ......T...
......A.-T TA..A.TG.. .C.......A T........C .T.--..... ...AG.....
...C.A...T .A........ ....A.A... ..C......C ....T.C..T .A....-.T.
.....A...T .A......CT .G....A... .........C ....T..... ........T.
.........T .A......CT .G....A... .........C ....T....T ........T.
.........T .A......CT .G....A... .........C ....T....T ........T.
.........T .A......CT .G....A... .........C ....T....T ........T.
.........T .A......CT .G....A... .........C ....T....T ........T.
.........T .A......CT .G....A... .........C ....T....T ........T.
...CAT...T GA..G..... .C..AT.CT. ....--AT.C C...G..... .....G..T.
...C.....T .A........ .C.A....T. ....--AT.C C...G..... ......T...
...C.....T .A........ .C.A....T. ....--AT.C C...G..... ......T...
AT.......A .G..G..... .C.T.T..AA T....CAT.C CA.CGA...A .A.AT..AT.
AT....A..A .G..G..... .TCC..A.AA .....CAT.C C..CGAT... .A..T..AT.
..TC.TA.-G .AC.A..... TC.A....T. ...TC..T.T .T.--....G .A..C..TTA
2、Length and variation of ITS1 and ITS2 of Dendrobium
Length(bp)
Gc content (%)
information site(%)
sequence divergence (%)
ITS1
228-233
43.8-55.6
41.01
20.47
ITS2
242-247
48.8-58.7
34.42
17.67
5.8S, 18S and 26S regions were all highly conserved. The
ITS1 and ITS2 regions were more variable, the inter-specific
sequence divergence is much larger than the intra-specific
variation between populations of same species .
 Conclusion：
Th results showed that differences between the various ITS
regions are big enough to differentiate each medicinal
Dendrobium species. Each Dendrobium species was found to
have a unique sequence in the ITS region, so that they could
be easily distinguished at the DNA level.
ITS could be used as molecular markers to distinguish the
Dendrobium species.
Part II:
Identification of Commercial Dried Caulis
Dendrobii by ITS sequencing
Materials: 22 group of
Huangcao and Fengdou
Shihu sliced crude drugs
(Yinpian) from different
location.
How to identify?
1. Extraction of total DNA from dried medicine
2. Amplification of ITS
3. Clone sequencing of ITS
4. Sequence blast with ITS from Genbank
Identification results of Huangcao shihu
Material
medicines
1.
Huangcao
2.
Huangcao
3.
Huangcao
4.
Huangcao
5.
Huangcao
6.
Huangcao
Location
Hanhui
Bozhou
Hanhui
Bozhou
Hanhui
Bozhou
Hanhui
Bozhou
Hanhui
Bozhou
Hanhui
Bozhou
Sequence blast result
Plant
Camparability
D. tosaense
636/638(99%)
Material
D. thyrsiflorum
616/617(99%)
D. thyrsiflorum
609/621(98%)
7.
Huangcao
D. nobile
636/636(100%)
D.ellipsophyllum
623/628(99%)
D. tosaense
635/636(99%)
D. aurantiacum
628/642(97%)
D. tosaense
633/636(99%)
D. nobile
636/636(100%)
D. nobile
570/581(98%)
D. tosaense
635/637(99%)
D. capillipes
628/628(100%)
D. capillipes
622/622(100%)
D. tosaense
Location
medicines
Sequence blast results
Plant
Heibei
Anguo
Comparability
D. nobile
636/636(100%)
D. tosaense
566/584(96%)
D. aurantiacum
626/635(98%)
D. tosaense
635/637(99%)
D. tosaense
D. acinaciforme
D. tosaense
D. nobile
D. linawianum
634/636(99%)
611/620(98%)
587/599(97%)
628/636(98%)
514/515(99%)
D. aurantiacum
D. tosaense
520/577(90%)
620/635(97%)
D. tosaense
D. linawianum
557/572(97%)
520/577(90%)
608/620 (98%)
D. tosaense
566/584(96%)
D. tosaense
648/668(97%)
D. thyrsiflorum
526/537(97%)
D. tosaense
D. ellipsophyllum
531/535(99%)
623/628(99%)
D. tosaense
628/628(100%)
D. capillipes
635/637(99%)
D. tosaense
564/567(99%)
D. chrysanthum
616/621(99%)
D. tosaense
D. nobile
549/557(98%)
636/636(100%)
D. tosaense
549/557(98%)
D. thyrsiflorum
D. tosaense
D. chrysanthum
D. tosaense
D. nobile
D. tosaense
D. tosaense
D. tosaense
609/621(98%)
514/515(99%)
631/636(99%)
577/591(97%)
570/581(98%)
529/544(97%)
566/584(96%)
529/544(97%)
8.
Huangcao
Shanghai
Huayu
9.
Huangcao
Jiangxi
Zhangshu
10.
Huangcao
Guangzhou
11.
Huangcao
Guangxi
Nanning
12.
Huangcao
Beijing
Heyanling
Brief summary： 9 Dendrobium species were identified, including D. tosaense, D.
thyrsiflorum, D. nobile, D. ellipsophyllum, D. aurantiacum, D. capillipes, D. linawianum, D.
chrysanthum, and D. acinaciforme from the twelve Huangcao samples.
Identification results of Fengdou shihu
Material
Medicines
1.
Fengdou
Location
Anhui
Bozhou
2.
Fengdou
Anhui
Bozhou
3
Fengdou
Anhui
Bozhou
4.
Fengdou
Anhui
Bozhou
5.
Fengdou
Anhui
Bozhou
Sequence blast results
Plant
comparability
D. tosaense
569/584 (97%)
D. pendulum
574/577 (99%)
D. tosaense
590/594 (99%)
D. falconeri
638/640 (99%)
D. tosaense
D. pendulum
D. falconeri
D. tosaense
D. chrysanthum
D. tosaense
635/637 (99%)
574/577 (99%)
629/640 (98%)
628/637 (98%)
631/636 (99%)
590/594 (99%)
D. tosaense
D. chrysanthum
D. pendulum
D .tosaense
590/592 (99%)
626/631 (99%)
567/577 (98%)
585/591 (98%)
D. tosaense
627/631 (99%)
D. officinale
628/637 (98%)
D. officinale
624/634 (98%)
D. tosaense
D. chrysanthum
D. chrysanthum
D. chrysanthum
456/468 (97%)
625/636 (98%)
622/631 (98%)
620/641 (96%)
Material
Location
Medicines
6.
Anhui
Fengdou
Bozhou
7.
Fengdou
Anhui
Bozhou
8.
Fengdou
Anhui
Bozhou
9.
Fengdou
Anhui
Bozhou
10.
Fengdou
Anhui
Bozhou
Sequenc blast results
Plant
Comparability
D. tosaense
635/636 (99%)
D. falconeri
633/635 (99%)
D. tosaense
447/447(100%)
D. chrysanthum 631/636 (99%)
D. tosaense
599/600 (99%)
D. tosaense
616/617 (99%)
D. tosaense
589/616 (95%)
D. officinale
631/635 (99%)
D. tosaense
553/557 (99%)
D. falconeri
635/640 (99%)
D. tosaense
585/591 (98%)
D. tosaense
628/630 (99%)
D. tosaense
632/636 (99%)
D. sinuatum
673/678 (99%)
D. tosaense
633/636 (99%)
D. tosaense
636/636 (100%)
D. officinale
631/635 (99%)
D. chrysanthum 620/634 (97%)
D. tosaense
625/636 (98%)
D. tosaense
627/636 (98%)
D. chrysanthum 590/590(100%)
D. tosaense
624/631 (98%)
D. officinale
631/636 (99%)
D. tosaense
635/636 (99%)
Brief summary： 6 Dendrobium species were identified, including D. tosaense, D.
pendulum, D. falconeri, D. chrysanthum, D. officinale, D. sinuatum from the ten
Fengdou samples.
Part III
Authentication of Commercial Dried Caulis
Dendrobii by dot blotting analysis
Induction:
•
Previously, we has succeeded in authentication the Dendrobium species of
commercial dried Shihu by sequences of ITS region. Now, we have further
established a
dot blotting as an effective method to authenticate the
source plant of Huangcao and Fengdou shihu.
•
It is very suitable, especially when a number of samples have to be
authenticated in a limited time.
How to identify?
• Preparation of hybridization membrane
Total DNA was extracted from several known
Dendrobium species and the corresponding ITS1-5.8SITS2 regions were amplified. Denatured DNA by 0.5 N
NaOH was spotted onto nitrocellulose membrane.
• Preparation of labelled probe:
Total DNA was extracted from commercial dried Shihu( to be
identified) and the ITS regions were amplified. Denatured these
products and incorporated with enzyme horseradish peroxidase.
•
Hybridization:
Carried out by a hybridization kit at 42 ℃ in a hybridization
oven.
•
Detection:
use the detection reagent to cover the blotting DNA and incubate,
then expose the blotting dot to the film, develop and fix image.
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