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1 Short ommunication Journal of piculture 33(3) : 221~226 (218) DOI: /apiculture Detection of Sugar ane (Saccharum officinarum)-specific Gene from Sugar and Sugar-honey younghee Kim, Somin Kim, Moonjung Kim, Jungmin Kim, Tai Truong and youngsu Yoon* Department of Life Science, ollege of Fusion Science, Kyonggi University (Received 31 ugust 218; Revised 21 September 218; ccepted 21 September 218) bstract Sugar cane-specific gene could be successfully amplified with DNs isolated from sugar or sugarhoney using Saccharum officinarum-specific Ultra-Rapid or conventional PR. Specificity of PR products from sugar or sugar-honey was verified by nested PR and DN sequencing. This PR could be applied to a quantitative analysis for honey-evaluation. In our knowledge, it is first report that sugar cane-specific sequence could be detected from sugar-honey or sugar itself, and that sugar-honey could be evaluated by genetic examination. Key words: Honey, Saccharum officinarum, Sugar cane, Sugar-honey, Sugar, Ultra-rapid PR odex EU, (O, 1995; EU ommission, 215),.,, (Yoo et al., 21). (MFDS, 214),. (Kim et al., 217). *orresponding author. bsyoon@kyonggi.ac.kr 221
2 222 Table 1. Specific primers information of Saccharum officinarum chloroplast gene Treatment primer PR product Reference Sequence (5 3 ) Molecular ane-cp-f GGTTTGGTTGTG loning* ane-cp-r TTGGGGT Detection ane-cp-df GTTTTTTTTTTGG PR ane-cp-dr GTTTGTGGTTT Nested ane-cp-nf TGGGTTTTGT PR ane-cp-nr GGTGGGTTTTTTTGTGTG *Primers for molecular cloning of Saccharum officinarum-specific matk gene on chloroplast. 698bp 271bp 137bp This study, E-IRMS (Elemental nalysis-isotope Ratio Mass Spectrometry) (Padovan et al., 23),,.,, 4, (Kim et al., 217).,,,. 1ppb (part per billion) (ODEX, 21), ppb 1gram 1ng., 1ng DN 3kb DN 1 8 ( ), PR 1 1 DN (, 217), PR., PR (Sobrino-Gregorio et al., 218),, (Laube et al., 21)..,.,,. (Sugar ane, Saccharum officinarum) maturase K (matk, Genbank accession No. LN849913). (matk) ( Sugar cane seeds;, ; 218.6). DN matk, DN pane-p-mat, (Table 1). DN, ( ; 1%; ; ) 8%(w/v), T (hexadecyltrimethyl ammonium bromide) DN-binding column (Qiagen, Germany). 1ml 2µl DN, 1µl PR. PR 1µl primer ( 1 pmole), 1µl DN,
3 Signal intensity 12 df/dt ycles Temperature (Degree) Fig. 1. mplification of S. officinarum-specific matk gene with DN from sugar. Panel. Fluorescence graph of S. officinarum-specific PRs with DNs from sugar cane seed (Seed), pane-p-mat (Positive), and samples 1, 2 (Sugar 1, 2) of sugar using primer, ane-cp-df/dr. Panel. Melting temperature analysis. Tms of PR products from sugar are estimated on and 75.88, respectively. Panel. garose gel electrophoresis of PR products. Lane M is DN size marker. Expected size of S. officinarum-specific PR product is 271 bp-long Signal intensity 12 df/dt ycles Temperature (Degree) Fig. 2. mplification of S. officinarum-specific matk gene with DN from sugar-honey. Panel. Fluorescence graph of S. officinarum-specific PRs with DNs from sugar cane seed (Seed), pane-p-mat (Positive), and samples 1, 2 (Honey 1, 2) of sugar-honey using primer, ane-cp-df/dr. Panel. Melting temperature analysis. Tms of PR products from sugar-honey are estimated on and 75.56, respectively. Panel. garose gel electrophoresis of PR products. Lane M is DN size marker. Expected size of S. officinarum-specific PR product is 271 bp-long. 5µl 2x Rapi:Detect Master mix (Genesystem o., Ltd., Korea), 2µl, 1µl/reaction. PR 95 3, 95, 3-55, 3-72, 3 1 cycle, 5 cycle GENEHEKER TM (Genesystem o., Ltd., Korea)., PR, DN pane-p-mat PR Tm, PR, DN (Fig. 1).
4 Signal intensity df/dt ycles Temperature (Degree) Fig. 3. mplification of S. officinarum-specific gene with DN from sugar-honey and sugar by using nested PR. Panel. Fluorescence graph of S. officinarum-specific nested PRs with DNs from sugar cane seed (Seed), pane-p-mat (Positive), and first PR product of sugarhoney (Honey 1, 2) and sugar (Sugar 1, 2) using primer, ane-cp-nf/nr. Panel. Melting temperature analysis. Panel. garose gel electrophoresis of nested PR products. Lane M is DN size marker. Expected size of S. officinarum-specific nested PR product is 137 bp-long. Fig. 4. lignment for sequences of PR products from each sample. DN sequences of the PR products from seed, sugar-honey and sugar were aligned. ll sequences were identical with matk (Genbank accession No. LN849913). (, 9%, 1%, 12 ), 1ml DN, PR, DN pane-p-mat. PR. DN pane-p-mat PR Tm PR, DN (Fig. 2). PR matk nested primer PR., detection primer 1 PR 1, nested PR, nested primer PR 1 PR., DN
5 225 Fig. 5. mplification of S. officinarum-specific matk gene with DN from seed, sugar-honey and sugar using conventional PR. Panel. Fluorescence graph of S. officinarum-specific PRs with DNs from sugar cane seed (Seed), pane-p-mat (Positive), sugar-honey (Honey 1, 2) and sugar (Sugar 1, 2) using primer, ane-cp-df/dr. Panel. Melting temperature analysis. Panel. garose gel electrophoresis of PR products. Lane M is DN size marker. Expected size of S. officinarum-specific PR product is 271 bp-long. matk PR 1 PR nested PR, pane-p-mat PR Tm DN, PR matk (Fig. 3). PR matk PR. PR (alignment analysis),,,, (pane-p-mat) PR, matk (Fig. 4)., PR PR PR. matk PR (S. officinarum-specific matk PR) PR, PR matk PR., PR ioneer Exicycler 96, PR ccupower 2X GreenStar qpr Master Mix (ioneer, Korea), PR primer (Table 1). PR 95 5, 95, 3-55, 3-72, 3 1 cycle, 4 cycle.,,,
6 226 DN PR, DN pane-p-mat PR Tm PR DN. matk PR (Fig. 5)., PR.,, (orn syrup), ( : PJ1482), ( ) 217 (No ).,, Truong Tai,, (ethina tumida). J. piculture 32: O Official Method of nalysis of O Intl. 2 th ed. Method ssociation of Official nalytical hemists, rlington, V, US. ODEX. 21. ODEX STNDRD FOR SUGRS. ODEX STN European ommission Official controls and enforcement. Honey russels, elgium. Kim, S. G., I. P. Hong, S. O. Woo, H. R. Jang, J. S. Jang and S. M. Han. 217a. hemical omposition of Korean Natural Honeys and Sugar Fed Honeys. The Korean Journal of Food and Nutrition 3: Laube, I., H. Hird, P. rodmann, S. Ullmann, M. Schöne- Michling, J. hisholm and H. roll. 21. Development of primer and probe sets for the detection of plant species in honey. Food hemistry 118: Lim, H. Y., S. H. Han and. S. Yoon Manual of Methods for Molecular iology : MFDS Food Standard ode. Ministry of Food and Drug Safety, heongju, Korea. Padovan, G. J., D. De Jong, L. P. Rodrigues and J. S. Marchini. 23. Detection of adulteration of commercial honey samples by the 13 / 12 isotopic ratio. Food hemistry 82: Sobrino-Gregorio, L., S. Vilanova, J. Prohens and I. Escriche Detection of honey adulteration by conventional and real-time PR. Food ontrol 95: Yoo, E.., Y. K. Kong and. S. Yoon. 21. Study on the Improved nalysis-methods to Determine dulterated Honeys. Korean Journal of piculture 25:
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