Int. J. Biol. Sci. 2009, 5 727 International Journal of Biological Sciences 2009; 5(7):727-735 © Ivyspring International Publisher. All rights reserved Research Paper Identification of Anoectochilus based on rDNA ITS sequences alignment and SELDI-TOF-MS Chuan Gao1, 3, Fusheng Zhang1, Jun Zhang4, Shunxing Guo1 , Hongbo Shao2,5 1. Institute of Medicinal Plant Development, Beijing Union Medical College/Chinese Academy of Medicinal Sciences, Beijing 100193, China; 2. Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling 712100, China; 3. Institute of Beijing Pharmacochemistry, Beijing 102205, China; 4. Central Laboratory of 306 Hospital of PLA, Beijing 100083, China; 5. Yantai Institute of Costal Zone Research, Chinese Academy of Sciences (CAS), Yantai 264003, China. Corresponding authors: [email protected] (Guo SX); [email protected] (Shao HB). Posting address: Dr. Professor Shao Hongbo, Yantai Institute of Costal Zone Research, Chinese Academy of Sciences (CAS), Yantai 264003, China. Received: 2009.08.28; Accepted: 2009.11.26; Published: 2009.12.02 Abstract The internal transcribed spacer (ITS) sequences alignment and proteomic difference of Anoectochilus interspecies have been studied by means of ITS molecular identification and surface enhanced laser desorption ionization time of flight mass spectrography. Results showed that variety certification on Anoectochilus by ITS sequences can not determine spe- cies, and there is proteomic difference among Anoectochilus interspecies. Moreover, pro- teomic finger printings of five Anoectochilus species have been established for identifying spe- cies, and genetic relationships of five species within Anoectochilus have been deduced ac- cording to proteomic differences among five species. Key words: Anoectochilus, ITS, proteomic finger printing, SELDI sterile condition. For a long time, the morphological 1. Introduction characteristics have been the main standards of phy- Anoectochilus is one of the 27 genera of subteib. logenetic studies. However, due to the narrow of the Goodyerinae of Trib. Neottieae (Orchidaceae). There are distribution of wild Anoectochilus and the scarce of about 40 species of this genus in tropical zone areas of natural reserves and the missing of flower period, Asia and Australia. 20 species among them and two traditional identification methods were usually inva- varieties have been found from southwest to south of lid. China [1]. Major medicinal plants named "King of With the development of plant molecular sys- medicine" or "Golden grass" of this genus are A. for- tematics, a growing number of nucleic acid analysis mosanus, A. koshunensis and A. roxburghii, which are techniques are widely used in plant phylogenetic generally used in treatment of hypertension, diabetes, studies [3], such as DNA hybridization, DNA restric- heart disease, pneumonia, acute and chronic hepati- tion enzyme spectrum analysis, DNA fingerprinting tis, nephritis and other diseases [1, 2]. techniques, RFLP, RAPD, AFLP and DNA sequence According to the identification standards [1], we analysis. At present, ITS sequence of ribosomal DNA collected wild species of Anoectochilus plants in (rDNA) has become an important molecular markers southwest areas of China and cultured axillary bud in http://www.biolsci.org Int. J. Biol. Sci. 2009, 5 728 in phylogenetic development of angiosperms, and The main instruments: Master Cycleer gradient been widely used in the intragenera, intergenera and AG 22331 PCR Instrument, Eppendorf companies; even interspecies phylogenetic studies [4-5]. A series protein flight mass spectrometer (PBS IIc), that is, of progress has been achieved [6-7]. surface-enhanced laser desorption ionization Meanwhile, proteomics researches based on time-of-flight mass spectrometry (SELDI-TOF), two-dimensional gel electrophoresis are also applied chemically modified weak-cation CM10 chip, Ci- in the genetic evolution and classification of species. phergen U.S. companies; Centrifuge 5810r desktop For instance, to identify the origin of cultivated spe- high-speed refrigerated centrifuge, Eppendorf com- cies through the analysis of protein differences of cul- pany; Milli-Q ultrapure water instrument, Millipore tivated and wild wheat [8], and to study the genetic products. diversity by protein differences of wheat varieties [9] 2.2 Methods or wheat groups planted in different areas [10]. In addition to wheat, other species used 2-D separated 2.2.1 Plant DNA extraction and amplification of ITS characteristic proteins to identify species (or subspe- sequences cies) , such as rice [11-12], sugarcane [13] and chilli Plant Genomic DNA Kit prospectus was a ref- [14], have been reported. erence to extract plant DNA. As a new way on proteome research, The primers of PCR amplification of ITS se- SELDI-TOF-MS (Surface Enhanced Laser Desorption quences [21, 22]: P1: CGT AAC AAG GTT TCC GTA Ionization-Time of Flight-Mass Spectrography) tech- GGT GAAC; P2: TTA TTG ATA TGC TTA AAC TCA nology shows a lot of advantages such as simple, fast, GCG GG, conventional PCR amplification conditions. acute and high-flux. It can detect trace protein as 2.2.2 Alignment of ITS sequences by Blast fmol(10-15mol) [15-16] and is regarded as supplement of two-dimensional electrophoresis. Today, SELDI Blast engine is in NCBI web site has been mostly used in clinical diagnosis through http://blast.ncbi.nlm.nih.gov/Blast.cgi. identifying several special proteins peaks of special 2.2.3 Sample preparation for proteomics disease [17] and also was used in the studies on plant When the sterile cultured plant seedlings grow proteome recently [18-20]. to 8-10cm high, they are collected from the tender In this experiment, based on the NCBI Blast shoots of plants and sample preparation is based on comparison of ITS sequences, several named (TW, FJ) the procedure described by Damerval [23], and modi- and unnamed (WS, YN1, YN2, XZ) orchid plants fied as follow: samples approximately 500 mg and 50 were only classified to genera. So the protein differ- mg polyvinylpyrrolidone(PVP) are ground in liquid ence of these plants using the SELDI-TOF-MS was nitrogen. The powder is suspended in 8-10 folds investigated to identify species at the first time, and sample volume (weight/vol) of acetone (–20 ºC) + the proteome fingerprint of these plants were estab- 10% vol/vol trichloroacetic acid (TCA) + 5% vol/vol lished respectively, and the genetic relationship of dithiothreitol (DTT) and incubated overnight at five species within Anoectochilus was preliminarily –20ºC. After centrifugation, (14, 000 × g, 4 ºC, 60 min), explored. the supernatant is removed, 5 mL of ice-cold acetone +1% vol/vol DTT+1 mM phenylmethyl sulfon- 2 Materials and methods ylfluoride (PMSF) is added, and the samples are stored for 1 h at –20 ºC. Then centrifuge at 4 ºC, 14, 2.1 Materials 000×g for 30 min, repeat this step one time. Volatilize Experimental material: six tissue culture seed- remains acetone of precipitation at -20 ºC. 20 mg pro- lings comprised TW (Taiwan province), FJ (Fujian tein powder is dissolved in 500 mL protein lysate (9.5 province), WS (Yunnan province), YN1(Yunnan mol/L urea, 2% 3-[(3-cholamidopropyl) dimethyl- province), YN2 (Yunnan province) and XZ (Tibet ammonio] -1-propanesulfonate (CHAPS), 1% DTT), province). insoluble particles are removed by centrifugation (14, The main reagents: polyvinylpyrrolidone (PVP), 000 ×g, 4 ºC, 30 min) and protein level is determined trichloroacetic acid (TCA), dithiothreitol (DTT), urea, using 2-D Quant Kit(Amersham) with accurate con- 3-[(3-cholamidopropyl)dimethylamonio]-1-propane centration using bovine serum albumin (BSA) as sulfonate (CHAPS) reagents for Gibco products; 2-D standard. Quant Kit protein quantitative kit products for the 2.2.4 SELDI-MS-TOF detection Amersham; Plant Genomic DNA Kit, TIANGEN company's products. Put the protein chip (Chemical modify chip CM10, Ciphergen company of USA) in processor. http://www.biolsci.org Int. J. Biol. Sci. 2009, 5 729 Add 200 µL binding buffer (50 mmol/L NaAc, pH 3 Result and Discussion 4.0) in each pore. Vibrate 2 times to wash the proces- 3.1 Results of alignment of ITS sequence by Blast sor at room temperature; 5 min every time, then shake away the buffer. Add 100 µL sample mixed The ITS sequences of samples were aligned by liquor in each pore (about 5μg protein per pore). Af- Blast. The four highest score DNA accession number ter Vibrating 1 h, shake away sample. Vibrate 2 times and their coverage and identity were listed in table 1. to wash processor with 200 μL elution buffer (50 TW, FJ, YN1, YN2, WS are classified in Anoectochilus, mmol/L NaAc, pH 4.0) at room temperature, 5 min TW could be judged as A. formosanus. Referring to its every time, then shake away the buffer. Wash again flower character, FJ can be identified as A. roxburghii. with deionized water, shake away immediately. Un- Although owning the same ITS sequences, YN1 and pack processor and take out chip, dry it in air, then YN2 may be two variants or subspecies because of its put 0.5 μL SPA [Sinapinic acid, 5 mg SPA in 80 μL differences in appearance. XZ belongs to Goodyera TFA and CAN solution (trifluoroacetic acid : acetoni- rather than Anoectochilus, can be identified as G. mac- trile=1:1) ] in each pore, after dry, repeat this step rantha. again then put chip in SELDI-TOF-MS instrument In table 1, Taxonomic status of the various gen- and detect (PBS IIc, Ciphergen company of USA). era and species are Eukaryota, Viridiplantae, Strep- tophyta, Embryophyta, Tracheophyta, Spermato- 2.2.5 Data processing and Proteins identification phyta, Magnoliophyta, Liliopsida, Asparagales, Or- Parameter set as: the detected highest molecular chidaceae, Orchidoideae, Cranichideae, Goodyerinae, weight is 50 kD, the optimizing range are 2-20 kilo under Goodyerinae are Anoectochilus, Ludisia, Dos- daltons (KD), the intensity of laser is 205, the detec- sinia, and Goodyera respectively. tion sensitivity is 8. The peaks under 2 kD are deleted The results also reveal that the classification re- to remove interference of base peak.