Botanical Studies (2007) 48: 239-242.
*
Corresponding author: E-mail: cxfu@zju.edu.cn; Phone:
+86-571-8820-6607; Fax: +86-571-8643-2273.
INTRODUCTION
"Cat ginseng" is one of the most commonly used
traditional Chinese medicines (TCM) for anti-tumor
therapy in East China (Jiangsu New Medicine College,
1984). It first attracted people¡¦s interest as a catnip
(Tucker and Tucker, 1988). Recently, three compounds,
i.e. dihydronepetalactone, iridomyrmecin, and
dihydroactinidiolide, have proven to be responsible for
the interesting response (Zhao et al., 2006a). Actinidia
valvata had been considered the source of "Cat ginseng"
before our recent studies confirmed A. macrosprma as
the genuine source (Zhao, 2006). It was not surprising
that A. macrosperma and A. valvata had been mistaken
for each other given the similarity in their morphological
and anatomical characters (Liang, 1984). A phylogenetic
analysis based on sequences of matK and ITS also
suggested that A. macrosperma and A. valvata were
sister species (Li et al., 2002). Besides A. valvata, two
more species (A. melanandra, A. chinensis) have been
misidentified as A. macrosperma, and their roots have also
been used as "cat ginseng" in China. The confusion may
have compromised the therapeutic value of this TCM and
jeopardized genuine resources for raw material production.
DNA markers, including PCR-RFLP and DNA
sequences, are useful for the identification and standard -
ization of TCM (Yang et al., 2001). The chloroplast trnD-
trnT region digested by endonuclease HinfI and DdeI has
shown a specific pattern in Sinopodophyllum hexandrum
that is distinct from species of Dysosma. We have used the
PCR-RFLP data to differentiate S. hexandrum (Gong et
al., 2006). The objective of this study was to differentiate
A. macrosperma from other Actinidia species using PCR-
RFLP data and DNA sequences of chloroplast trnK region.
MATERIALS AND METHODS
Seventeen samples were used in this study representing
ten species and four sections of Actinidia (Table 1), and
their voucher specimens are deposited in the Herbarium of
Zhejiang University (HZU). DNAs were extracted from
silica-gel dried leaves using a modified CTAB method
(Doyle, 1991). PCR (polymerase chain reaction) was con-
ducted using primers W83040 (5¡¦-GGG TTG CCC GGG
ACT CGA AC-3¡¦) and W83041 (5¡¦-CAA CGG TAG AGT
ACT CGG CTT TTA-3¡¦) for trnK (Demesure et al., 1995).
A 50 £gl PCR amplification run contained 50 ng template
DNA, 5 £gl 10¡Ñbuffer, 2 mM MgCl
2
, 200 £gM dNTPs,
0.4 £gM of each primer and 2.0 U Taq DNA polymerase.
The PCR reaction was performed using a PTC-100 PCR
DNA Thermal Cycler (Bio-Rad, USA), and the cycling
program included an initial 4 min denaturation at 94¢XC,
which was followed by 35 amplification cycles with 1
min denaturation at 94¢XC, 1 min annealing at 65¢XC, and
a 1.5 min extension at 72¢XC. A final extension step of 7
Authentication of Actinidia macrosperma using PCR-
RFLP based on trnK sequences
Yun-Peng ZHAO, Ying-Xiong QIU, Wei GONG, Jian-Hua LI, and Cheng-Xin FU*
Research Program for Resource Botany and Phytochemistry, Lab. of Systematic & Evolutionary Botany and Biodiversity,
College of Life Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
(Received September 1, 2006; Accepted December 14, 2006)
ABSTRACT
. "Cat ginseng", the dried root of Actinidia macrosperma, is a famous traditional Chinese
medicine against cancers in eastern China. The roots of some other species of the genus Actinidia such as A.
valvata and A. melanandra have also been used as fake "cat ginseng", but they have little therapeutic value.
However, identification of the original plants used to make the crude drugs is difficult, especially during the
vegetation period. In this study we developed molecular markers for the determination and authentication of
A. macrosperma. The restriction digestion of the chloroplast trnK region using endonucleases DdeI and DraI
produces two unique patterns in A. macrosperma, and in the matK sequences, there are 11 sites unique to A.
macrosperma. The molecular markers provide an effective and accurate identification and authentication of A.
macrosperma in Actinidia.
Keywords: Actinidia macrosperma; Actinidia valvata; PCR-RFLP; Molecular marker.
MOLECULAR BIOLOgy
