Identifying Pulsatilla Chinesis (Bunge) Regel, and Potentilla Discolor

Bunge from Each Other using Restrictive Enzyme Dde I

Erjia Wang

, Shaoxuan Zhang

2,*

, Dejun Sun

, Guangzhu Lin

and Tiantian Wang

Li Ying Clinic of Combination with Traditional Chinese and Western Medicine, #6366 Nanhu Rd., Changchun, China

Laboratory of Molecular Genetics, Department of Advanced Biomedical Techniques, Institute of Frontier Medical

Sciences, Jilin University, #1163 Xinmin St., Changchun, China

Laboratory of Molecular Drugs, Department of Advanced Biomedical Techniques, Institute of Frontier Medical Sciences,

Jilin University, #1163 Ximin St., Changchun, China

Cardiovascular Disease Diagnosis and Treatment Centre, The First Hospital of Jilin University, #71 Xinmin St.,

Changchun, China

Keywords: Dde I, Identification, Potentilla discolor Bunge, Pulsatilla chinesis (Bunge) Regel.

Abstract: For identifying Pulsatilla chinesis (Bunge) Regel, and Potentilla discolor Bunge from each other, a new

method was established. We amplified the ITS regions of them, and sequenced the purified PCR products

directly. We edited and compared the obtained sequences by Genetyx and BioEdit. The possible sites of

restriction endonucleases were searched using PREMIER 5.0. It was found that Dde I can be used for their

identification. In this case, we concluded that Dde I can be used effectively in identification of these plants.

1 INTRODUCTION

Because of its reliable therapeutic effects, Pulsatilla

chinesis (Bunge) Regel, was included in

Pharmacopoeia of The People's Republic of China,

almost every edition (Chinese Pharmacopoeia

Commisson, 2015). Potentilla discolor Bunge, has

also the similar effects, but it was not included in

Pharmacopoeia of The People's Republic of China

since before. Currently, it was found to hold the

function of curing diabetes, so it was started to be

included in Pharmacopoeia of The People's Republic

of China (Chinese Pharmacopoeia Commisson, 2015).

With the lack of knowledge and morphological

similarity, some regions in China still use them as

each other

(New Medical College of Jiangsu, 1985),

affecting the collecting and even the correct usage,

and more importantly, the therapeutic effects of the

two materia medica. So how to identify the two

materia medica becomes very important. Although

researchers had created and even improved some

methods such as those based on appearance,

differences in structure under microscopy and

differences in chemical components (Zhang et al.,

2000), a thin-layer chromatography (Liu and Lei,

2005), but due to the similarities, it is hard to identify

them from each other precisely with these methods.

With the advance in molecular biology,

authentication and identification using molecular

biology techniques (Tang and Fu, 2000) becomes

more and more popular in decades (Wang, 2001).

And above all, they are very reliable. So, in this study,

based on the fundamental techniques of molecular

biology, we established a new method to identify

Pulsatilla chinesis and Potentilla discolour.

2 MATERIALS AND METHODS

2.1 Plants

We collected Pulsatilla chinesis nearby Changchun,

China. Potentilla discolor was purchased from

YAODUBAICAOYANGSHENGTANG. Pulsatilla

chinesis was authenticated by Professor Minglu Deng

of Changchun University of Traditional Chinese

Medicine and Potentilla discolor, Wenchang Guo of

Jilin University (Table 1). We dried the plants with

silica gel and preserved them in our laboratory. The

leaves were used for the experiments.

Wang, E., Zhang, S., Sun, D., Lin, G. and Wang, T.

Identifying Pulsatilla Chinesis (Bunge) Regel, and Potentilla Discolor Bunge from Each Other using Restrictive Enzyme Dde I.

DOI: 10.5220/0011594800003430

In Proceedings of the 8th International Conference on Agricultural and Biological Sciences (ABS 2022), pages 29-31

ISBN: 978-989-758-607-1; ISSN: 2795-5893

Table 1: Geological, purchasing information and the dates

of sample collection.

Plants Geological or purchasing

information

Dates

Pulsatilla

chinesis

(Bunge)

egel

Collected at No.027 Country

Road (4KM far from Tuding

Town, Shuangyang,

Changchun)

2013-

6-2

Potentilla

discolor

Purchased from

YAODUBAICAOYANGSH

ENGTANG

2013-

3-25

2.2 DNA Preparation

We took a small amount of leaf from every sample

and cleaned them with cotton swabs to eliminate the

impurities on the leaves with 70% alcohol in a culture

dish. Then we dried the cleaned leaves at room

temperature for a while and grinded them into

powders with liquid nitrogen. We collected the

powders and prepared genome DNA using Plant

DNA Isolation Reagents (Takara Biotechnology)

following the provider’s instruction. The qualities of

extracted DNA were checked in 1% agarose slab gels.

2.3 Primers

The primers of ITS reported in a former research

(Takaiwa et al., 1985) were selected and synthesized

by Takara Biotechnology Co., Ltd (Dalian, China).

2.4 PCR Reactions

Every PCR reaction was performed following the

former research (Takaiwa et al., 1985) in a total

amount of 50μL [1μL, each of the primers in 2.3, 5μL

every genome DNA, 5μL Reaction Buffer,

5μLdNTPs, 1μL Taq DNA polymerase (Takara

Biotechnology Co. Ltd)]. The PCR conditions are as

follows: 94℃, 1 cylce,5 min, 35 cycles (denature at

94℃,1 min; annealing at 55℃ 2 min; extension at

72℃,2 min), 72℃, 1 cycle for 10 min. MiniCycler

PTC-150(MJ Research Inc,) was used to perform the

PCR reactions. PCR products were checked in 1%

agarose slab gels.

2.5 Sequencing

2.4 PCR products were purified using PCR Filter

Units (Millipore Corporation) then directly

sequenced. We performed the sequencing reactions in

a 10μL mixture for each sample using ABI BigDye

Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems) with every sequencing primer. The

sequencing reactions conditions are as follows: 96℃,

1 cycle for 1 min, 25 cycles (denature at 96℃,30 sec,

extension 50℃, 5 sec.), 60℃, 1 cycle, 4 min. We

analysed the obtained sequences using 3130

Sequencer (Applied Biosystems.).

2.6 Comparisons and Editings

We used Genetyx-SV/RC version 11.0 and BioEidt

version 7.0.9 to edit and compare the sequences.

2.7 Searching for Appropriate Restriction

Endonuclease

We used Primer PREMIER (version 5.0, PREMIER

Biosoft international, CA, USA) to search for a

restrictive site that can be used, eventually, we found

that Dde I can digest the two PCR products in

different sites and can be recruited to identify them

from each other.

2.8 Dde I Digestion

We used the purified the 2.4 PCR products for

digestions. Dde I digestions were performed at 37℃

for 2h. We performed the reactions in MiniCycler

PTC-150. Dde I was purchased from Biolabs (New

England Biolabs.). We confirmed the digestions in a

2% agarose gel.

Figure 1: Differences in ITS sequences of Pulsatilla

chinesis and Potentilla discolor.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

Notes: Left to right, 50bp Marker; Pulsatilla chinesis;

Potentilla discolor.

Figure 2: DdeI digestion.

3 RESULTS

Table 1 shows the geological, purchasing information

and the dates. For each species, three different

individual plants were put into use for sequencing.

Figure 1. shows the differences of ITS sequences

in two plants.

Electrophoresis of the digests (Figure 2).

4 CONCLUSIONS AND

DISCUSSION

We for the first time sequenced and reported

Pulsatilla chinesis (Zhang et al. 2017) and Potentilla

discolor ITS sequences (Zhang et al. 2015). Using

these sequences, we established a new simple method

to identify them from each other, that can ensure the

correct use of those two drugs, especially in case they

are used to cure different disease (for example,

diabetes). As shown above, the new method we

created in this study (first amplifying the ITS regions

and then digesting them with Dde I) is very simple

and reliable, so even a kit for identification is

reasonable. It can used in the procedures such as

acquisition, quality control etc. of the rude drugs.

Although, further experimentation and confirmation

are necessary.

Although we can use ITS sequences themselves

directly to identify these two plants. But sequencing

itself is a complicated technique and it need

expensive equipments, like sequencer, to conduct the

experiments. So, this new method should be a more

practical one to be put in use.

Pulsatilla chinesis and Potentilla discolor, both

have wide distributions in China, so differences in

samples of different area can be predicted. For precise

identification, enlargement of analysis in samples of

different area and accumulation of knowledge are

necessary.

REFERENCES

Chinese Pharmacopoeia Commission, (2015).

Pharmacopoeia of The People's Republic of China,

China Medical Science Press, Beijing, 2015 edition,

pages. 371-372.

Chinese Pharmacopoeia Commission, (2015).

Pharmacopoeia of The People's Republic of China,

China Medical Science Press, Beijing, 2015 edition,

pages.380-381.

Liu, W. X., and Lei, G. L. (2005). Application of DNA

molecular diagnosis techniques in identification of

traditional Chinese medicine. Journal of Shaanxi

College of Traditional Medicine. (28):30-32

New Medical College of Jiangsu, (1985). A Dictionary of

Traditional Chinese Medicine, Shanghai Science and

Technology Press, Shanghai. 1

edition. pages.704-

706. (in Chinese).

Takaiwa, F., Oono, K., and Sugiura, M. (1985). Nucleotide

sequence of the 17s-25s spacer region from rice rDNA.

Plant Molecular Biology. 4:355-364.

Tang, S. Y., and Fu, W. (2000). The development of new

biological techniques drives the innovation in the

identification of traditional Chinese medicine. West

China Journal of Pharmaceutical Sciences. (15):115-

116 (in Chinese).

Wang, S. Q. (2001). Identification of Pulsatilla chinesis

from its most used varieties. Lishizhen Medicine and

Materia Medica Research. (12):428-429 (in Chinese).

Zhang, D. L., Fu, X., and Yang, Y. Y. (2000). Identification

of Pulsatilla chinesis and its varietues of confusion.

Chinese Traditional and Herbal Drugs. (31):554-556

(in Chinese).

Zhang, S. X., Lin, G.Z., Wang, T.T., and Li, Y.R. (2017),

ITS and trnL-F sequences analysis of Pulsatilla

chinensis (Bge.) Regal. Advances in Biological

Sciences Research. 4:254-257.

Zhang, S. X., Li, Y. R., Wang, T. T., Lin, G. Z., and Wang,

B. C. (2015). ITS and trnL-F sequences analysis of

Potentilla discolour Bge. 3

International Conference

on Material, Mechanical and Manufacturing

Engineering (IC3ME). pages.469-472.

Identifying Pulsatilla Chinesis (Bunge) Regel, and Potentilla Discolor Bunge from Each Other using Restrictive Enzyme Dde I