KR20110131667A - Differentially expressed p-psbb genes in wild ginseng and discrimination method of wild ginseng using the same - Google Patents
Differentially expressed p-psbb genes in wild ginseng and discrimination method of wild ginseng using the same Download PDFInfo
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Abstract
Description
본 발명은 산삼에서 발현된 특이 유전자에 관한 것으로, 보다 상세하게는 억제적 공제 하이브리다이제이션(suppressive subtraction hybridization; SSH)법을 이용하여 산삼으로부터 발현되는 특이 유전자인 엽록체 p-psbB(Panax ginseng chloroplast psbB, 이하 "p-psbB"라 칭함)을 분리해 내고, 이를 이용하여 산삼을 감별해낼 수 있도록 한 산삼에서 발현된 특이 유전자 p-psbB 및 이를 이용한 산삼의 감별방법에 관한 것이다.The present invention relates to a specific gene expressed in wild ginseng, and more particularly, chloroplast p-psbB (Panax ginseng chloroplast psbB), which is a specific gene expressed from wild ginseng using a suppressive subtraction hybridization (SSH) method. The present invention relates to a specific gene p-psbB expressed in wild ginseng and a method for discriminating wild ginseng using the same, to separate wild ginseng using the same.
인삼(Panax ginseng C.A. Meyer)은 두릅나무과에 속하는 낙엽성 다년생 식물이고, 인삼속으로 현재 12종이 확인된 것으로, 수천년간 중국, 일본, 한국을 포함하여 동양에서 널리 사용되는 약초중의 하나이다. 이러한 인삼의 속명은 그리스어인 '파낙스(Panax)'로, '만병통치약'이라는 뜻에서 유래되었으며, 약초의 뿌리가 사람과 같은 형상을 갖고 있어 인삼으로 불리워지고 있다. Ginseng (Panax ginseng C.A. Meyer) is a deciduous perennial plant belonging to the family Arboraceae, and 12 species of ginseng have been identified, one of the herbs widely used in the East including China, Japan, and Korea for thousands of years. The generic name of ginseng is Panax, which is derived from the meaning of panacea, and the root of the herb is called ginseng.
이와 같은 인삼은 특히 호르몬, 심장혈관, 면역, 중추신경계에서 긍정적인 효과를 나타내고, 피로의 예방, 산화손상, 인공 돌연변이, 암 예방 등을 포함하여 건강에 이로운 특성을 나타내고 있다. 이러한 인삼의 약학적 효과는 대부분 다양한 인삼사포닌(ginsenoside)에 의한 것으로, 특히 트리터페노이드 사포닌(triterpenoid saponin) 성분에서 기인하며, 다양한 인삼사포닌의 생리학적 약학 효과는 다르고, 심지어 반대될 수도 있다.Such ginseng has a particularly beneficial effect on hormones, cardiovascular system, immunity, central nervous system, and has beneficial health properties including fatigue prevention, oxidative damage, artificial mutation, cancer prevention, and the like. The pharmaceutical effects of these ginsengs are mostly caused by various ginseng saponins (ginsenosides), in particular due to the triterpenoid saponin component, and the physiological and pharmaceutical effects of various ginseng saponins are different and may even be reversed.
한편, 인삼은 재배장소 및 방법에 따라 재배된 인삼, 야생산삼, 산양산삼으로 분류될 수 있다. 상기 재배된 인삼은 인공적으로 배양된 품종으로 현재 시장의 대부분을 차지하고 있다. 또한 상기 야생산삼은 깊은 산의 자연환경에서 자라는 것이고, 산양산삼은 숲이나 산에 씨를 뿌려 자라는 것으로 야생산삼의 모조품으로 고려될 수 있다.On the other hand, ginseng can be classified into ginseng, wild ginseng, mountain goats grown in accordance with the place and method of cultivation. The cultivated ginseng is an artificially cultivated variety and currently occupies most of the market. In addition, the wild ginseng is to grow in the natural environment of the deep mountain, Sanyangsan ginseng can be considered as a counterfeit of wild ginseng to grow by seeding the forest or mountain.
이와 같은 재배된 인삼, 야생산삼, 산양산삼은 모두 건강에 유익한 효력을 지니고 있기는 하나, 재배된 인삼과 산삼 사이에는 인삼사포닌 성분에 미차가 있는 것으로 보고되었으며, 한국과 중국에서는 재배된 인삼보다 산삼이 더 건강에 유익한 효력을 갖는 것으로 널리 인정받고 있다.Although these grown ginseng, wild ginseng, and wild ginseng have all the beneficial effects on health, there is a reported difference in ginseng saponin components between the grown ginseng and wild ginseng. It is widely recognized as having a more beneficial effect on health.
이에 본 발명자들은 산삼으로부터 억제적 공제 하이브리다이제이션(Suppressive subtraction hybridzation; SSH)법을 이용하여 재배인삼에서는 발현되지 않고, 산삼에서만 특이하게 발현되는 유전자를 분리해내었으며, 이에 따라 상기 특이 유전자를 이용할 경우 산삼을 감별해낼 수 있음을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors have isolated a gene that is not expressed in cultivated ginseng but specifically expressed in wild ginseng using Suppressive subtraction hybridzation (SSH) from wild ginseng. It was confirmed that wild ginseng can be discriminated and completed the present invention.
이를 위하여 본 발명은 산삼에서 특이하게 발현되어 산삼을 감별하는데 유용하게 사용될 수 있는 특이 유전자 p-psbB를 제공하는데 그 목적이 있다.To this end, it is an object of the present invention to provide a specific gene p-psbB which is specifically expressed in wild ginseng that can be usefully used to discriminate wild ginseng.
또한 본 발명은 상기 유전자 p-psbB를 이용하여 산삼을 감별하는 방법을 제공하는데 다른 목적이 있다.Another object of the present invention is to provide a method for discriminating wild ginseng using the gene p-psbB.
본 발명은 상기와 같은 목적을 달성하기 위하여The present invention to achieve the above object
산삼에서 특이하게 발현되어 서열번호 3의 염기서열을 갖는 것을 특징으로 하는 산삼에서 발현된 특이 유전자 p-psbB를 제공한다.It provides a specific gene p-psbB expressed in wild ginseng characterized by having a nucleotide sequence of SEQ ID NO: 3 specifically expressed in wild ginseng.
또한, 본 발명은 시료를 실시간 정량 RT-PCR 하는 단계와;In addition, the present invention comprises the steps of real-time quantitative RT-PCR sample;
서열번호 3의 염기서열을 갖는 p-psbB 유전자를 마커로 하고 상기 RT-PCR 생성물을 확인하여 상기 시료에서 p-psbB 유전자의 검출여부를 확인하는 단계를 포함하는 것을 특징으로 하는 유전자 p-psbB를 이용한 산삼의 감별방법을 제공한다.P-psbB gene having a nucleotide sequence of SEQ ID NO: 3 as a marker and the RT-PCR product by identifying the p-psbB gene characterized in that it comprises the step of detecting the p-psbB gene in the sample Provides a differentiation method of wild ginseng used.
또한, 상기 시료를 RT-PCR하는 단계는 시료인 인삼 또는 산삼으로부터 전체 RNA를 분리하는 단계와; 서열번호 4에 나타낸 염기서열로 이루어진 정방향 프라이머 및 서열번호 5에 나타낸 염기서열로 이루어진 역방향 프라이머를 이용하여 cDNA를 합성하는 단계와; 상기 cDNA를 주형으로 PCR 증폭하는 단계;를 포함하는 것을 특징으로 한다.In addition, the step of RT-PCR of the sample comprises the steps of separating the total RNA from the ginseng or wild ginseng as a sample; Synthesizing cDNA using a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 4 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 5; PCR amplifying the cDNA into a template; characterized in that it comprises a.
또한, 상기 RT-PCR 생성물의 확인은 2%의 아가로스겔에서 전기영동하는 방법이나, RT-PCR 생성물을 60℃에서 90℃로 0.5℃씩 온도를 증가시켜 측정하는 용융커브 분석법을 통해 이루어지는 것을 특징으로 한다.In addition, the identification of the RT-PCR product is performed by electrophoresis on agarose gel of 2%, or through a melt curve analysis to measure the RT-PCR product by increasing the temperature by 0.5 ℃ from 90 ℃ to 90 ℃ It features.
상술한 바와 같은 본 발명에 따른 산삼에서 발현되는 p-psb유전자는 인삼에서는 발현되지 않으면서 산삼에서는 특이하게 발현되기 때문에 이를 마커로 사용하면 산삼의 진품여부를 감별하는데 유용하게 사용될 수 있고, 더 나아가 산삼의 품질을 보존 및 발전시키는 시스템의 확보나 산삼의 사업화에 적절하게 사용될 수 있다.Since the p-psb gene expressed in wild ginseng according to the present invention as described above is specifically expressed in wild ginseng without being expressed in ginseng, it can be usefully used to discriminate the authenticity of wild ginseng when used as a marker. It can be used appropriately to secure the system to preserve and develop the quality of wild ginseng and to commercialize wild ginseng.
도 1은 SSH의 공정을 개략적으로 나타낸 개요도.
도 2는 p-psbB유전자의 실시간 정량 RT-PCR 분석결과를 나타낸 도면.
도 3은 p-psbB의 특이적인 발현의 semi-quantitative RT-PCR 분석결과를 나타낸 도면.1 is a schematic diagram schematically showing a process of SSH.
Figure 2 shows the real-time quantitative RT-PCR analysis of the p-psbB gene.
Figure 3 shows the results of semi-quantitative RT-PCR analysis of specific expression of p-psbB.
이하에서는 본 발명에 대하여 보다 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in more detail.
본 발명에서는 산삼에서 특이하게 발현되는 서열번호 3의 염기서열을 갖는 특이 유전자 p-psbB를 제공한다.The present invention provides a specific gene p-psbB having a nucleotide sequence of SEQ ID NO: 3 specifically expressed in wild ginseng.
산삼에서 특이하게 발현되는 p-psbB 유전자를 확인하기 위하여 당해분야에서 통상적으로 사용되는 억제적 공제 하이브리다이제이션(Suppressive subtraction hybridization, SSH)법을 이용하였다.In order to identify p-psbB genes that are specifically expressed in wild ginseng, Suppressive subtraction hybridization (SSH) was commonly used in the art.
도 1은 SSH법을 개략적으로 나타낸 도면으로서, 도 1에 도시된 바와 같이 산삼의 cDNA에서 인삼의 cDNA와 공통되는 부분을 제거하여 공통된 cDNA가 상당히 감소되도록 하여 산삼의 특이 cDNA가 풍부해졌다. SSH를 통해 100이상의 라이브러리가 얻어졌고, colony-PCR에 의해 검출된 재조합효율은 약 90%였다. 그리고 PCR 증폭에 의해 확인된 100개의 양성클론들은 16개의 상당히 다른 클론으로부터 랜덤하게 서열을 이루었다. 그중 본 발명에서는 신규한 유전자인 p-psbB를 분리했다.FIG. 1 schematically illustrates the SSH method. As shown in FIG. 1, the cDNA of wild ginseng removes a part common to the cDNA of ginseng so that the common cDNA is significantly reduced, thereby enriching the specific cDNA of wild ginseng. More than 100 libraries were obtained via SSH, and the recombination efficiency detected by colony-PCR was about 90%. And 100 positive clones identified by PCR amplification were randomly sequenced from 16 significantly different clones. Among them, the present invention isolated p-psbB, a novel gene.
염기서열 분석을 통해 산삼에서 특이적으로 발현되는 신규한 유전자 p-psbB는 서열번호 3의 염기서열을 갖는 것을 확인하였다.The sequencing analysis confirmed that the novel gene p-psbB specifically expressed in wild ginseng has the nucleotide sequence of SEQ ID NO: 3.
본 발명에 따른 p-psbB 유전자가 산삼에서 특이적으로 발현되는 것을 확인하기 위하여 통상의 방법에 따라 실시간 정량 RT-PCR을 분석하였다. RT-PCR분석에는 5개의 재배인삼과 2개의 산삼의 전체 RNA를 사용하였으며, cDNA를 증폭하기 위하여 하기 표 1에 나타낸 p-psbB 유전자 특이 프라이머를 설계하여 사용하였다. 그리고 각 RT-PCR반응에서 동등한 양의 cDNA가 사용된 것을 확인하기 위하여 18s 프라이머를 대조구로 사용하였다.In order to confirm that p-psbB gene according to the present invention is specifically expressed in wild ginseng, real-time quantitative RT-PCR was analyzed according to a conventional method. RT-PCR analysis was used for the total RNA of five cultivated ginseng and two wild ginseng, and to design a p-psbB gene specific primer shown in Table 1 to amplify the cDNA. And 18s primer was used as a control to confirm that the equivalent amount of cDNA was used in each RT-PCR reaction.
R: 5'-CCT GTC GGC CAA GGT TAT AG-3'F: 5'-AAC GAG ACC TCA GCC TGC TA-3 '
R: 5'-CCT GTC GGC CAA GGT TAT AG-3 '
R: 5'-TGT CTT AAC GAG CGG GAA TC-3' (서열번호 5)F: 5'-TGT CTT AAC GAG CGG GAA TC-3 '(SEQ ID NO: 4)
R: 5'-TGT CTT AAC GAG CGG GAA TC-3 '(SEQ ID NO: 5)
그 결과 도 2에 보여지는 바와 같이 산삼에서 p-psbB 유전자의 상대적인 양이 상당히 높음을 확인할 수 있었으며, 재배인삼에서는 p-psbB 전사가 거의 검출되지 않은 것을 알 수 있다.As a result, as shown in Figure 2 it was confirmed that the relative amount of the p-psbB gene in wild ginseng is considerably high, it can be seen that almost no p-psbB transcription in cultivated ginseng.
한편, 본 발명에 따른 p-psbB 유전자의 특이적인 발현을 좀 더 확인하기 위하여 통상의 방법에 따라 semi-quantitative RT-PCR분석을 실시하였다. 4개의 재배인삼 및 2개의 산삼의 전체 RNA를 RT-PCR분석에 사용했다. 약 200개의 염기상의 cDNA 조각을 증폭하기 위하여 상기 표 1에 나타낸 p-psbB 유전자 특이 프라이머를 설계하여 사용하였다. 각 RT-PCR 반응에서 동등한 양의 RNA가 사용된 것을 확인하기 위하여 18s 프라이머들을 대조구로 사용하였다. 그리고, PCR 생성물의 세기는 18s로 관찰된 PCR 생성물 세기와 대조하여 표준화 했다. PCR 생성물은 아가로스겔의 농도스캐닝에 의해 정량화하였다. On the other hand, semi-quantitative RT-PCR analysis was performed according to a conventional method in order to further confirm the specific expression of the p-psbB gene according to the present invention. Total RNA from four cultivated ginseng and two wild ginseng was used for RT-PCR analysis. The p-psbB gene specific primers shown in Table 1 above were designed and used to amplify cDNA fragments on about 200 bases. 18s primers were used as controls to confirm that equal amounts of RNA were used in each RT-PCR reaction. The PCR product intensity was normalized against the PCR product intensity observed at 18 s. PCR products were quantified by concentration scanning of agarose gels.
그 결과 도 3에 도시된 바와 같이 p-psbB 유전자는 재배인삼에 비하여 산삼에서 상당히 높게 발현되는 것을 알 수 있었다.
As a result, as shown in Figure 3, the p-psbB gene was found to be significantly higher in wild ginseng than in cultivated ginseng.
상기와 같은 결과들을 토대로 하여 볼 때 p-psbB 유전자는 인삼에서는 특이적으로 발현되지 않고, 산삼에서는 특이적으로 발현되는 것을 알 수 있다. 따라서 상기한 p-psbB유전자는 산삼을 감별하는데 중요한 마커로 사용될 수 있다.Based on the above results, it can be seen that the p-psbB gene is not specifically expressed in ginseng, but specifically in wild ginseng. Therefore, the p-psbB gene may be used as an important marker for discriminating wild ginseng.
따라서 본 발명에서는 시료를 RT-PCR하는 단계와, 본 발명에 따라 산삼에서 분리된 p-psbB유전자를 마커로 하여 상기 RT-PCR 생성물을 확인하여 상기 시료에서 p-psbB 유전자가 검출되었는지의 여부를 확인하는 단계;를 포함하는 산삼의 감별방법을 제공한다.Therefore, in the present invention, the RT-PCR of the sample and the p-psbB gene isolated from wild ginseng according to the present invention as a marker identify the RT-PCR product to determine whether the p-psbB gene is detected in the sample. It provides a method of differentiating wild ginseng, including the step of identifying.
그리고, 상기 RT-PCR 생성물의 확인은 2%의 아가로스겔에서 전기영동하는 방법이나, RT-PCR 생성물을 60℃에서 90℃로 0.5℃씩 온도를 증가시켜 측정하는 용융커브 분석법을 통해 이루어질 수 있다.In addition, the identification of the RT-PCR product may be performed by electrophoresis on 2% agarose gel or through a melt curve analysis method in which the RT-PCR product is measured by increasing the temperature by 0.5 ° C from 60 ° C to 90 ° C. have.
이하 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다.
Hereinafter, preferred examples are provided to help understanding of the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.
<실시예 1> 전체 RNA 분리 및 mRNA 정제Example 1 Total RNA Isolation and mRNA Purification
5개의 재배인삼과 2개의 산삼으로부터 RNeasy Plant RNA Isolation kit(Qiagen)을 이용하여 제조사의 사용설명서에 기재된 방법으로 전체 RNA를 분리하였다. 분리된 RNA의 농도는 260㎚에서 흡광도로 측정하였으며, RNA 추출물은 제조사의 사용설명서에 기재된 방법으로 Superscript Ⅲ 역전사 효소(Invirtogen)와 랜덤 핵사머를 사용하여 cDNA를 합성하기 전에 제한효소인 DNase-Ⅰ(Invitrogen)으로 절단하여 사용하였다.
Total RNA was isolated from five cultivated ginseng and two wild ginseng using the RNeasy Plant RNA Isolation kit (Qiagen) by the method described in the manufacturer's instructions. The concentration of the isolated RNA was measured by absorbance at 260 nm, and RNA extract was prepared using the restriction enzyme DNase-Ⅰ before synthesis of cDNA using Superscript III reverse transcriptase (Invirtogen) and random nucleomers by the method described in the manufacturer's instructions. It was cut into (Invitrogen) and used.
<실시예 2> 억제적 공제 하이브리다이제이션(Suppressive subtractive hybridization, SSH)Example 2 Suppressive subtractive hybridization (SSH)
실시예 1에서 분리된 산삼과 재배인삼의 전체 RNA를 사용하여 SSH를 실시하였다. SSH는 Clontech PCR-Select™ cDNA Subtraction Kit(Clontech)를 이용하여 제조사의 프로토콜에 따라 실시하였다. SSH로부터 유도한 공제 라이브러리(tester: 산삼, driver: 인삼) cDNA 조각을 pEC-T vector(KOMA Co., Seoul, Korea)로 삽입하였다. 삽입된 조각을 포함하는 양성 클론은 colony-PCR을 통해 확인하였다.
SSH was performed using the total RNA of wild ginseng and cultivated ginseng isolated in Example 1. SSH was performed according to the manufacturer's protocol using the Clontech PCR-Select ™ cDNA Subtraction Kit (Clontech). A derivation library (tester: wild ginseng, driver: ginseng) cDNA fragments derived from SSH were inserted into the pEC-T vector (KOMA Co., Seoul, Korea). Positive clones containing the inserted fragments were identified by colony-PCR.
<실시예 3> 실시간 정량 RT-PCRExample 3 Real-Time Quantitative RT-PCR
유전자 특이 프라이머를 사용하여 SSH 라이브러리에서 특이하게 발현되는 유전자를 비교하기 위하여 StepOne machine과 Fast SYBR Green Master Mix(Biosystem. USA)를 이용하여 실시간 정량 RT-PCR을 실시하였다. 이때 RT-PCR을 위해 사용된 유전자 특이 프라이머는 표 1과 같다.Real-time quantitative RT-PCR was performed using a StepOne machine and Fast SYBR Green Master Mix (Biosystem. USA) to compare genes specifically expressed in SSH libraries using gene specific primers. The gene specific primers used for RT-PCR are shown in Table 1.
세포로부터 추출된 전체 RNA 2㎍에서 First Strand cDNA Synthesis kit(invitrogen)를 이용하여 cDNA 40㎕로 역전사하였다. 각 실시간 정량 RT-PCR 반응은 10㎕의 2 X Fast SYBR Green Master Mix, 0.4μM 의 정방향 프라이머(서열번호 4)와 0.4μM의 역방향 프라이머(서열번호 5), 2.75㎕의 DNase-free H2O, 1.5㎕ cDNA 주형을 포함하는 20㎕의 반응 혼합물에서 실시하였다. PCR증폭은 95℃에서 8분간 실시하고, 40사이클의 증폭(95℃에서 45초, 56℃에서 45초, 72℃에서 45초)을 실시하였다. 상기 PCR생성물을 60℃에서 95℃로 0.5℃씩 점차적으로 증가시키면서 녹였다. 증폭산물의 동일성과 반응의 특이성은 용융커브 분석법을 통해 확인하였으며, 3회 반복실험으로 측정하였으며, 그 결과는 도 2에 나타내었다. 이때 internal control로서 18s를 사용하였다.
2 μg of total RNA extracted from the cells was reverse transcribed into 40 μl of cDNA using a First Strand cDNA Synthesis kit (invitrogen). Each real-time quantitative RT-PCR reaction consisted of 10 μl of 2 X Fast SYBR Green Master Mix, 0.4 μM of forward primer (SEQ ID NO: 4) and 0.4 μM of reverse primer (SEQ ID NO: 5), 2.75 μl of DNase-free H 2 O And 20 μl of reaction mixture containing 1.5 μl cDNA template. PCR amplification was performed at 95 ° C for 8 minutes, and 40 cycles of amplification (45 seconds at 95 ° C, 45 seconds at 56 ° C, 45 seconds at 72 ° C) were performed. The PCR product was dissolved in increments of 0.5 ° C from 60 ° C to 95 ° C. The identity of the amplification product and the specificity of the reaction were confirmed by melt curve analysis, measured in three replicates, and the results are shown in FIG. 2. 18s was used as internal control.
<실시예 4> semi-quantitative RT-PCRExample 4 semi-quantitative RT-PCR
유전자 특이 프라이머를 사용하여 SSH 라이브러리에서 특이하게 발현되는 유전자를 비교하기 위하여 Semi-quantitative RT-PCR을 실시하였다. Semi-quantitative RT-PCR was performed to compare genes specifically expressed in SSH libraries using gene specific primers.
cDNA를 합성하기 위하여 실시예 1에서 분리한 4개의 재배인삼 및 2개의 산삼의 전체 RNA 2g을 First strand cDNA Synthesis kit (invitrogen)에 따라 사용했고, 1.0㎕의 cDNA를 PCR을 위한 주형으로 사용하였다. PCR 증폭은 95℃에서 5분, 95℃에서 45초, 54℃에서 30초, 72℃에서 60를 한 사이클로 하여 총 30사이클을 반복수행하였다. 최종적으로 72℃에서 5분 동안 반응시켰다. PCR 생성물은 2% 아가로스겔에서 전기영동하였으며, 그 결과는 도 3에 나타내었다. 이때 대조구로는 GAPDH mRNA를 사용하였다.
To synthesize cDNA, 2 g of total RNA of four cultivated ginseng and two wild ginseng isolated in Example 1 were used according to First strand cDNA Synthesis kit (invitrogen), and 1.0 μl of cDNA was used as a template for PCR. PCR amplification was repeated 5 cycles at 95 ° C., 45 seconds at 95 ° C., 30 seconds at 54 ° C., and 60 cycles at 72 ° C. for a total of 30 cycles. Finally, the reaction was carried out at 72 ° C. for 5 minutes. PCR products were electrophoresed on 2% agarose gel and the results are shown in FIG. 3. At this time, GAPDH mRNA was used as a control.
<실시예 5> Sequencing and homologyExample 5 Sequencing and homology
PCR생성물은 pEC-T vector(KOMA CO., LTd, Korea)에 삽입한 다음, ABI 3700 DNA sequencers(Perkin-Elmer Applied Biosystems)를 이용하여 염기서열을 확인하였다. 염기서열 분석에 사용된 소프트웨어는 Chromas sequence analysis software를 이용하여 수행하였고, 분석된 전체 염기서열을 서열번호 1로 기재하고, 서열번호 1로부터 암호화되는 아미노산 서열을 서열번호 2에 기재하였으며, 전체 염기서열 중에서도 단백질로 발현되는 코딩부분의 염기서열만을 서열번호 3에 나타내었다.
PCR products were inserted into the pEC-T vector (KOMA CO., LTd, Korea), and then sequenced using ABI 3700 DNA sequencers (Perkin-Elmer Applied Biosystems). The software used for sequencing was performed using Chromas sequence analysis software, the entire sequence analyzed was set forth in SEQ ID NO: 1, the amino acid sequence encoded from SEQ ID NO: 1 is set forth in SEQ ID NO: 2, and the entire nucleotide sequence Among them, only the nucleotide sequence of the coding part expressed by the protein is shown in SEQ ID NO: 3.
상기와 같이 본 발명에 따른 p-psbB 유전자는 서열번호 3의 염기서열을 가지고 있음을 확인할 수 있었으며, 상기한 p-psbB 유전자는 도 2, 도 3에 나타나는 바와 같이 인삼에 비하여 산삼에서 상당히 높게 발현되는 것을 확인할 수 있었다. As described above, it was confirmed that the p-psbB gene according to the present invention has the nucleotide sequence of SEQ ID NO: 3, and the p-psbB gene is significantly higher in wild ginseng than in ginseng as shown in FIGS. 2 and 3. It could be confirmed.
따라서 본 발명에 따른 p-psbB 유전자는 산삼을 판별하는데 중요한 마커로 사용될 수 있음을 확인할 수 있다.
Therefore, it can be seen that the p-psbB gene according to the present invention can be used as an important marker for discriminating wild ginseng.
CG: 재배인삼
WG, MWG: 산삼CG: Growing Ginseng
WG, MWG: wild ginseng
<110> Sangji University Industry academic cooperation foundation <120> Differentially expressed p-psbB genes in wild ginseng and discrimination method of wild ginseng using the same <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1527 <212> DNA <213> Artificial Sequence <220> <223> Differentially expressed p-psbB genes in wild ginseng <220> <221> CDS <222> (1)..(1524) <400> 1 atg ggt ttg cct tgg tat cgt gtt cat acc gtt gta ttg aat gat ccc 48 Met Gly Leu Pro Trp Tyr Arg Val His Thr Val Val Leu Asn Asp Pro 1 5 10 15 ggc cgg ttg ctt tct gtc cat ata atg cat aca gct cta gtt gct ggt 96 Gly Arg Leu Leu Ser Val His Ile Met His Thr Ala Leu Val Ala Gly 20 25 30 tgg gcg ggt tcc atg gct cta tat gaa tta gca gtt ttt gat ccc tct 144 Trp Ala Gly Ser Met Ala Leu Tyr Glu Leu Ala Val Phe Asp Pro Ser 35 40 45 gac cct gtt ctt gat cca atg tgg aga cag ggt atg ttc gtt ata ccc 192 Asp Pro Val Leu Asp Pro Met Trp Arg Gln Gly Met Phe Val Ile Pro 50 55 60 ttc atg act cgt tta gga ata acc aat tca tgg ggc ggt tgg agt gtc 240 Phe Met Thr Arg Leu Gly Ile Thr Asn Ser Trp Gly Gly Trp Ser Val 65 70 75 80 aca gga ggg gct ata ccg aat ccg ggt att tgg agt tac gaa ggt gtg 288 Thr Gly Gly Ala Ile Pro Asn Pro Gly Ile Trp Ser Tyr Glu Gly Val 85 90 95 gcc ggg gca cat att gtg ttt tct ggc ttg tgc ttc ttg gca gct atc 336 Ala Gly Ala His Ile Val Phe Ser Gly Leu Cys Phe Leu Ala Ala Ile 100 105 110 tgg cat tgg gtt tat tgg gat cta gaa att ttt tct gat gaa cgt aca 384 Trp His Trp Val Tyr Trp Asp Leu Glu Ile Phe Ser Asp Glu Arg Thr 115 120 125 gga aaa cct tct ttg gat ttg ccc aag atc ttt gga att cat tta ttt 432 Gly Lys Pro Ser Leu Asp Leu Pro Lys Ile Phe Gly Ile His Leu Phe 130 135 140 ctc gca ggg gtg gct tgc ttt ggt ttt ggt gca ttt cat gta aca ggc 480 Leu Ala Gly Val Ala Cys Phe Gly Phe Gly Ala Phe His Val Thr Gly 145 150 155 160 ttg tat ggt cct gga ata tgg gtg tct gat cct tat gga cta acg gga 528 Leu Tyr Gly Pro Gly Ile Trp Val Ser Asp Pro Tyr Gly Leu Thr Gly 165 170 175 aaa gta caa tct gta aat cca gcg tgg ggt gtg gaa ggt ttt gat cct 576 Lys Val Gln Ser Val Asn Pro Ala Trp Gly Val Glu Gly Phe Asp Pro 180 185 190 ttt gtt ccg gga gga ata gcc tct cat cat att gca gca ggg aca ttg 624 Phe Val Pro Gly Gly Ile Ala Ser His His Ile Ala Ala Gly Thr Leu 195 200 205 ggt ata ttg gcc ggt cta ttc cat ctt agt gtc cgc ccg ccc caa cgt 672 Gly Ile Leu Ala Gly Leu Phe His Leu Ser Val Arg Pro Pro Gln Arg 210 215 220 cta tac aaa gga ttg cgt atg ggc aat att gaa acc gtc ctt tcc agt 720 Leu Tyr Lys Gly Leu Arg Met Gly Asn Ile Glu Thr Val Leu Ser Ser 225 230 235 240 agt atc gct gct gtc ttt ttt gca gct ttt gtt gtt gcc gga act atg 768 Ser Ile Ala Ala Val Phe Phe Ala Ala Phe Val Val Ala Gly Thr Met 245 250 255 tgg tat ggt tca gca act acc ccg atc gaa tta ttt ggg cct act cgt 816 Trp Tyr Gly Ser Ala Thr Thr Pro Ile Glu Leu Phe Gly Pro Thr Arg 260 265 270 tat caa tgg gat cag ggg tac ttc cag caa gag ata tat cga aga gtt 864 Tyr Gln Trp Asp Gln Gly Tyr Phe Gln Gln Glu Ile Tyr Arg Arg Val 275 280 285 agt gct ggg cta gcc gaa aat caa agt tta tca gaa gct tgg tct aaa 912 Ser Ala Gly Leu Ala Glu Asn Gln Ser Leu Ser Glu Ala Trp Ser Lys 290 295 300 att cct gaa aaa tta gct ttt tat gat tac atc ggc aat aat ccg gca 960 Ile Pro Glu Lys Leu Ala Phe Tyr Asp Tyr Ile Gly Asn Asn Pro Ala 305 310 315 320 aaa ggg gga tta ttc aga gcg ggt tca atg gat aac ggg gat gga ata 1008 Lys Gly Gly Leu Phe Arg Ala Gly Ser Met Asp Asn Gly Asp Gly Ile 325 330 335 gcg gtt gga tgg tta gga cat cct atc ttt aga gat aaa gaa ggg cgt 1056 Ala Val Gly Trp Leu Gly His Pro Ile Phe Arg Asp Lys Glu Gly Arg 340 345 350 gaa ctt ttt gta cgt cgt atg cct acc ttt ttt gaa acc ttt ccg gtc 1104 Glu Leu Phe Val Arg Arg Met Pro Thr Phe Phe Glu Thr Phe Pro Val 355 360 365 gtt ttg gta gat ggc gac gga att gtt aga gcc gat gtt cct ttt cga 1152 Val Leu Val Asp Gly Asp Gly Ile Val Arg Ala Asp Val Pro Phe Arg 370 375 380 agg gca gaa tcg aag tat agt gtc gaa caa gta ggt gta act gtt gag 1200 Arg Ala Glu Ser Lys Tyr Ser Val Glu Gln Val Gly Val Thr Val Glu 385 390 395 400 ttc tac ggc ggc gaa ctc aac gga gtc agt tat agt gat cct gct act 1248 Phe Tyr Gly Gly Glu Leu Asn Gly Val Ser Tyr Ser Asp Pro Ala Thr 405 410 415 gtg aaa aaa tat gct aga cgt gct caa ttg ggt gaa att ttt gaa tta 1296 Val Lys Lys Tyr Ala Arg Arg Ala Gln Leu Gly Glu Ile Phe Glu Leu 420 425 430 gat cgt gct act ttg aaa tcc gat ggt gtt ttt cgt agc agt ccg agg 1344 Asp Arg Ala Thr Leu Lys Ser Asp Gly Val Phe Arg Ser Ser Pro Arg 435 440 445 ggt tgg ttt act ttt gga cat gct tct ttt gct ttg ctc ttc ttt ttt 1392 Gly Trp Phe Thr Phe Gly His Ala Ser Phe Ala Leu Leu Phe Phe Phe 450 455 460 gga cat att tgg cat ggt gct aga acc ttg ttc aga gat gtt ttt gct 1440 Gly His Ile Trp His Gly Ala Arg Thr Leu Phe Arg Asp Val Phe Ala 465 470 475 480 ggt att gat cca gat ttg gat gct caa gta gaa ttt gga gca ttc caa 1488 Gly Ile Asp Pro Asp Leu Asp Ala Gln Val Glu Phe Gly Ala Phe Gln 485 490 495 aaa ctg gga gat cca act aca aga aga caa gta gtc tga 1527 Lys Leu Gly Asp Pro Thr Thr Arg Arg Gln Val Val 500 505 <210> 2 <211> 508 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Leu Pro Trp Tyr Arg Val His Thr Val Val Leu Asn Asp Pro 1 5 10 15 Gly Arg Leu Leu Ser Val His Ile Met His Thr Ala Leu Val Ala Gly 20 25 30 Trp Ala Gly Ser Met Ala Leu Tyr Glu Leu Ala Val Phe Asp Pro Ser 35 40 45 Asp Pro Val Leu Asp Pro Met Trp Arg Gln Gly Met Phe Val Ile Pro 50 55 60 Phe Met Thr Arg Leu Gly Ile Thr Asn Ser Trp Gly Gly Trp Ser Val 65 70 75 80 Thr Gly Gly Ala Ile Pro Asn Pro Gly Ile Trp Ser Tyr Glu Gly Val 85 90 95 Ala Gly Ala His Ile Val Phe Ser Gly Leu Cys Phe Leu Ala Ala Ile 100 105 110 Trp His Trp Val Tyr Trp Asp Leu Glu Ile Phe Ser Asp Glu Arg Thr 115 120 125 Gly Lys Pro Ser Leu Asp Leu Pro Lys Ile Phe Gly Ile His Leu Phe 130 135 140 Leu Ala Gly Val Ala Cys Phe Gly Phe Gly Ala Phe His Val Thr Gly 145 150 155 160 Leu Tyr Gly Pro Gly Ile Trp Val Ser Asp Pro Tyr Gly Leu Thr Gly 165 170 175 Lys Val Gln Ser Val Asn Pro Ala Trp Gly Val Glu Gly Phe Asp Pro 180 185 190 Phe Val Pro Gly Gly Ile Ala Ser His His Ile Ala Ala Gly Thr Leu 195 200 205 Gly Ile Leu Ala Gly Leu Phe His Leu Ser Val Arg Pro Pro Gln Arg 210 215 220 Leu Tyr Lys Gly Leu Arg Met Gly Asn Ile Glu Thr Val Leu Ser Ser 225 230 235 240 Ser Ile Ala Ala Val Phe Phe Ala Ala Phe Val Val Ala Gly Thr Met 245 250 255 Trp Tyr Gly Ser Ala Thr Thr Pro Ile Glu Leu Phe Gly Pro Thr Arg 260 265 270 Tyr Gln Trp Asp Gln Gly Tyr Phe Gln Gln Glu Ile Tyr Arg Arg Val 275 280 285 Ser Ala Gly Leu Ala Glu Asn Gln Ser Leu Ser Glu Ala Trp Ser Lys 290 295 300 Ile Pro Glu Lys Leu Ala Phe Tyr Asp Tyr Ile Gly Asn Asn Pro Ala 305 310 315 320 Lys Gly Gly Leu Phe Arg Ala Gly Ser Met Asp Asn Gly Asp Gly Ile 325 330 335 Ala Val Gly Trp Leu Gly His Pro Ile Phe Arg Asp Lys Glu Gly Arg 340 345 350 Glu Leu Phe Val Arg Arg Met Pro Thr Phe Phe Glu Thr Phe Pro Val 355 360 365 Val Leu Val Asp Gly Asp Gly Ile Val Arg Ala Asp Val Pro Phe Arg 370 375 380 Arg Ala Glu Ser Lys Tyr Ser Val Glu Gln Val Gly Val Thr Val Glu 385 390 395 400 Phe Tyr Gly Gly Glu Leu Asn Gly Val Ser Tyr Ser Asp Pro Ala Thr 405 410 415 Val Lys Lys Tyr Ala Arg Arg Ala Gln Leu Gly Glu Ile Phe Glu Leu 420 425 430 Asp Arg Ala Thr Leu Lys Ser Asp Gly Val Phe Arg Ser Ser Pro Arg 435 440 445 Gly Trp Phe Thr Phe Gly His Ala Ser Phe Ala Leu Leu Phe Phe Phe 450 455 460 Gly His Ile Trp His Gly Ala Arg Thr Leu Phe Arg Asp Val Phe Ala 465 470 475 480 Gly Ile Asp Pro Asp Leu Asp Ala Gln Val Glu Phe Gly Ala Phe Gln 485 490 495 Lys Leu Gly Asp Pro Thr Thr Arg Arg Gln Val Val 500 505 <210> 3 <211> 297 <212> DNA <213> Artificial Sequence <220> <223> Differentially expressed p-psbB genes in wild ginseng <400> 3 cctgatccca ttgataacga gtaggcccaa ataattcgat cagggtagtt gctgaaccat 60 accacatagt tccggcaaca acaaaagctg caaaaaagac agcagcgata ctactggaaa 120 ggacggtttc aatattgccc atacgcaatc ctttgtatag acgttggggc gggcggacac 180 taagatggaa tagaccggcc aatataccca atgtccctgc tgcaatatga tgagaggcta 240 ttcctcccgg aacaaaagga tcaaaacctt ccacacccca cgctggattt acagatt 297 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RT-PCR forward primer <400> 4 tgtcttaacg agcgggaatc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RT-PCR reverse primer <400> 5 tgtcttaacg agcgggaatc 20 <110> Sangji University Industry academic cooperation foundation <120> Differentially expressed p-psbB genes in wild ginseng and discrimination method of wild ginseng using the same <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1527 <212> DNA <213> Artificial Sequence <220> <223> Differentially expressed p-psbB genes in wild ginseng <220> <221> CDS (222) (1) .. (1524) <400> 1 atg ggt ttg cct tgg tat cgt gtt cat acc gtt gta ttg aat gat ccc 48 Met Gly Leu Pro Trp Tyr Arg Val His Thr Val Val Leu Asn Asp Pro 1 5 10 15 ggc cgg ttg ctt tct gtc cat ata atg cat aca gct cta gtt gct ggt 96 Gly Arg Leu Leu Ser Val His Ile Met His Thr Ala Leu Val Ala Gly 20 25 30 tgg gcg ggt tcc atg gct cta tat gaa tta gca gtt ttt gat ccc tct 144 Trp Ala Gly Ser Met Ala Leu Tyr Glu Leu Ala Val Phe Asp Pro Ser 35 40 45 gac cct gtt ctt gat cca atg tgg aga cag ggt atg ttc gtt ata ccc 192 Asp Pro Val Leu Asp Pro Met Trp Arg Gln Gly Met Phe Val Ile Pro 50 55 60 ttc atg act cgt tta gga ata acc aat tca tgg ggc ggt tgg agt gtc 240 Phe Met Thr Arg Leu Gly Ile Thr Asn Ser Trp Gly Gly Trp Ser Val 65 70 75 80 aca gga ggg gct ata ccg aat ccg ggt att tgg agt tac gaa ggt gtg 288 Thr Gly Gly Ala Ile Pro Asn Pro Gly Ile Trp Ser Tyr Glu Gly Val 85 90 95 gcc ggg gca cat att gtg ttt tct ggc ttg tgc ttc ttg gca gct atc 336 Ala Gly Ala His Ile Val Phe Ser Gly Leu Cys Phe Leu Ala Ala Ile 100 105 110 tgg cat tgg gtt tat tgg gat cta gaa att ttt tct gat gaa cgt aca 384 Trp His Trp Val Tyr Trp Asp Leu Glu Ile Phe Ser Asp Glu Arg Thr 115 120 125 gga aaa cct tct ttg gat ttg ccc aag atc ttt gga att cat tta ttt 432 Gly Lys Pro Ser Leu Asp Leu Pro Lys Ile Phe Gly Ile His Leu Phe 130 135 140 ctc gca ggg gtg gct tgc ttt ggt ttt ggt gca ttt cat gta aca ggc 480 Leu Ala Gly Val Ala Cys Phe Gly Phe Gly Ala Phe His Val Thr Gly 145 150 155 160 ttg tat ggt cct gga ata tgg gtg tct gat cct tat gga cta acg gga 528 Leu Tyr Gly Pro Gly Ile Trp Val Ser Asp Pro Tyr Gly Leu Thr Gly 165 170 175 aaa gta caa tct gta aat cca gcg tgg ggt gtg gaa ggt ttt gat cct 576 Lys Val Gln Ser Val Asn Pro Ala Trp Gly Val Glu Gly Phe Asp Pro 180 185 190 ttt gtt ccg gga gga ata gcc tct cat cat att gca gca ggg aca ttg 624 Phe Val Pro Gly Gly Ile Ala Ser His His Ile Ala Ala Gly Thr Leu 195 200 205 ggt ata ttg gcc ggt cta ttc cat ctt agt gtc cgc ccg ccc caa cgt 672 Gly Ile Leu Ala Gly Leu Phe His Leu Ser Val Arg Pro Pro Gln Arg 210 215 220 cta tac aaa gga ttg cgt atg ggc aat att gaa acc gtc ctt tcc agt 720 Leu Tyr Lys Gly Leu Arg Met Gly Asn Ile Glu Thr Val Leu Ser Ser 225 230 235 240 agt atc gct gct gtc ttt ttt gca gct ttt gtt gtt gcc gga act atg 768 Ser Ile Ala Ala Val Phe Phe Ala Ala Phe Val Val Ala Gly Thr Met 245 250 255 tgg tat ggt tca gca act acc ccg atc gaa tta ttt ggg cct act cgt 816 Trp Tyr Gly Ser Ala Thr Thr Pro Ile Glu Leu Phe Gly Pro Thr Arg 260 265 270 tat caa tgg gat cag ggg tac ttc cag caa gag ata tat cga aga gtt 864 Tyr Gln Trp Asp Gln Gly Tyr Phe Gln Gln Glu Ile Tyr Arg Arg Val 275 280 285 agt gct ggg cta gcc gaa aat caa agt tta tca gaa gct tgg tct aaa 912 Ser Ala Gly Leu Ala Glu Asn Gln Ser Leu Ser Glu Ala Trp Ser Lys 290 295 300 att cct gaa aaa tta gct ttt tat gat tac atc ggc aat aat ccg gca 960 Ile Pro Glu Lys Leu Ala Phe Tyr Asp Tyr Ile Gly Asn Asn Pro Ala 305 310 315 320 aaa ggg gga tta ttc aga gcg ggt tca atg gat aac ggg gat gga ata 1008 Lys Gly Gly Leu Phe Arg Ala Gly Ser Met Asp Asn Gly Asp Gly Ile 325 330 335 gcg gtt gga tgg tta gga cat cct atc ttt aga gat aaa gaa ggg cgt 1056 Ala Val Gly Trp Leu Gly His Pro Ile Phe Arg Asp Lys Glu Gly Arg 340 345 350 gaa ctt ttt gta cgt cgt atg cct acc ttt ttt gaa acc ttt ccg gtc 1104 Glu Leu Phe Val Arg Arg Met Pro Thr Phe Phe Glu Thr Phe Pro Val 355 360 365 gtt ttg gta gat ggc gac gga att gtt aga gcc gat gtt cct ttt cga 1152 Val Leu Val Asp Gly Asp Gly Ile Val Arg Ala Asp Val Pro Phe Arg 370 375 380 agg gca gaa tcg aag tat agt gtc gaa caa gta ggt gta act gtt gag 1200 Arg Ala Glu Ser Lys Tyr Ser Val Glu Gln Val Gly Val Thr Val Glu 385 390 395 400 ttc tac ggc ggc gaa ctc aac gga gtc agt tat agt gat cct gct act 1248 Phe Tyr Gly Gly Glu Leu Asn Gly Val Ser Tyr Ser Asp Pro Ala Thr 405 410 415 gtg aaa aaa tat gct aga cgt gct caa ttg ggt gaa att ttt gaa tta 1296 Val Lys Lys Tyr Ala Arg Arg Ala Gln Leu Gly Glu Ile Phe Glu Leu 420 425 430 gat cgt gct act ttg aaa tcc gat ggt gtt ttt cgt agc agt ccg agg 1344 Asp Arg Ala Thr Leu Lys Ser Asp Gly Val Phe Arg Ser Ser Pro Arg 435 440 445 ggt tgg ttt act ttt gga cat gct tct ttt gct ttg ctc ttc ttt ttt 1392 Gly Trp Phe Thr Phe Gly His Ala Ser Phe Ala Leu Leu Phe Phe Phe 450 455 460 gga cat att tgg cat ggt gct aga acc ttg ttc aga gat gtt ttt gct 1440 Gly His Ile Trp His Gly Ala Arg Thr Leu Phe Arg Asp Val Phe Ala 465 470 475 480 ggt att gat cca gat ttg gat gct caa gta gaa ttt gga gca ttc caa 1488 Gly Ile Asp Pro Asp Leu Asp Ala Gln Val Glu Phe Gly Ala Phe Gln 485 490 495 aaa ctg gga gat cca act aca aga aga caa gta gtc tga 1527 Lys Leu Gly Asp Pro Thr Thr Arg Arg Gln Val Val 500 505 <210> 2 <211> 508 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Leu Pro Trp Tyr Arg Val His Thr Val Val Leu Asn Asp Pro 1 5 10 15 Gly Arg Leu Leu Ser Val His Ile Met His Thr Ala Leu Val Ala Gly 20 25 30 Trp Ala Gly Ser Met Ala Leu Tyr Glu Leu Ala Val Phe Asp Pro Ser 35 40 45 Asp Pro Val Leu Asp Pro Met Trp Arg Gln Gly Met Phe Val Ile Pro 50 55 60 Phe Met Thr Arg Leu Gly Ile Thr Asn Ser Trp Gly Gly Trp Ser Val 65 70 75 80 Thr Gly Gly Ala Ile Pro Asn Pro Gly Ile Trp Ser Tyr Glu Gly Val 85 90 95 Ala Gly Ala His Ile Val Phe Ser Gly Leu Cys Phe Leu Ala Ala Ile 100 105 110 Trp His Trp Val Tyr Trp Asp Leu Glu Ile Phe Ser Asp Glu Arg Thr 115 120 125 Gly Lys Pro Ser Leu Asp Leu Pro Lys Ile Phe Gly Ile His Leu Phe 130 135 140 Leu Ala Gly Val Ala Cys Phe Gly Phe Gly Ala Phe His Val Thr Gly 145 150 155 160 Leu Tyr Gly Pro Gly Ile Trp Val Ser Asp Pro Tyr Gly Leu Thr Gly 165 170 175 Lys Val Gln Ser Val Asn Pro Ala Trp Gly Val Glu Gly Phe Asp Pro 180 185 190 Phe Val Pro Gly Gly Ile Ala Ser His His Ile Ala Ala Gly Thr Leu 195 200 205 Gly Ile Leu Ala Gly Leu Phe His Leu Ser Val Arg Pro Pro Gln Arg 210 215 220 Leu Tyr Lys Gly Leu Arg Met Gly Asn Ile Glu Thr Val Leu Ser Ser 225 230 235 240 Ser Ile Ala Ala Val Phe Phe Ala Ala Phe Val Val Ala Gly Thr Met 245 250 255 Trp Tyr Gly Ser Ala Thr Thr Pro Ile Glu Leu Phe Gly Pro Thr Arg 260 265 270 Tyr Gln Trp Asp Gln Gly Tyr Phe Gln Gln Glu Ile Tyr Arg Arg Val 275 280 285 Ser Ala Gly Leu Ala Glu Asn Gln Ser Leu Ser Glu Ala Trp Ser Lys 290 295 300 Ile Pro Glu Lys Leu Ala Phe Tyr Asp Tyr Ile Gly Asn Asn Pro Ala 305 310 315 320 Lys Gly Gly Leu Phe Arg Ala Gly Ser Met Asp Asn Gly Asp Gly Ile 325 330 335 Ala Val Gly Trp Leu Gly His Pro Ile Phe Arg Asp Lys Glu Gly Arg 340 345 350 Glu Leu Phe Val Arg Arg Met Pro Thr Phe Phe Glu Thr Phe Pro Val 355 360 365 Val Leu Val Asp Gly Asp Gly Ile Val Arg Ala Asp Val Pro Phe Arg 370 375 380 Arg Ala Glu Ser Lys Tyr Ser Val Glu Gln Val Gly Val Thr Val Glu 385 390 395 400 Phe Tyr Gly Gly Glu Leu Asn Gly Val Ser Tyr Ser Asp Pro Ala Thr 405 410 415 Val Lys Lys Tyr Ala Arg Arg Ala Gln Leu Gly Glu Ile Phe Glu Leu 420 425 430 Asp Arg Ala Thr Leu Lys Ser Asp Gly Val Phe Arg Ser Ser Pro Arg 435 440 445 Gly Trp Phe Thr Phe Gly His Ala Ser Phe Ala Leu Leu Phe Phe Phe 450 455 460 Gly His Ile Trp His Gly Ala Arg Thr Leu Phe Arg Asp Val Phe Ala 465 470 475 480 Gly Ile Asp Pro Asp Leu Asp Ala Gln Val Glu Phe Gly Ala Phe Gln 485 490 495 Lys Leu Gly Asp Pro Thr Thr Arg Arg Gln Val Val 500 505 <210> 3 <211> 297 <212> DNA <213> Artificial Sequence <220> <223> Differentially expressed p-psbB genes in wild ginseng <400> 3 cctgatccca ttgataacga gtaggcccaa ataattcgat cagggtagtt gctgaaccat 60 accacatagt tccggcaaca acaaaagctg caaaaaagac agcagcgata ctactggaaa 120 ggacggtttc aatattgccc atacgcaatc ctttgtatag acgttggggc gggcggacac 180 taagatggaa tagaccggcc aatataccca atgtccctgc tgcaatatga tgagaggcta 240 ttcctcccgg aacaaaagga tcaaaacctt ccacacccca cgctggattt acagatt 297 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> RT-PCR forward primer <400> 4 tgtcttaacg agcgggaatc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> RT-PCR reverse primer <400> 5 tgtcttaacg agcgggaatc 20
Claims (4)
서열번호 3의 염기서열을 갖는 p-psbB 유전자를 마커로 하고 상기 RT-PCR 생성물을 확인하여 상기 시료에서 p-psbB 유전자의 검출여부를 확인하는 단계를 포함하는 것을 특징으로 하는 유전자 p-psbB를 이용한 산삼의 감별방법.Real time quantitative RT-PCR of the sample;
P-psbB gene having a nucleotide sequence of SEQ ID NO: 3 as a marker and the RT-PCR product by identifying the p-psbB gene characterized in that it comprises the step of detecting the p-psbB gene in the sample Differentiation method of wild ginseng used.
상기 시료를 RT-PCR하는 단계는 시료인 인삼 또는 산삼으로부터 전체 RNA를 분리하는 단계와;
서열번호 4에 나타낸 염기서열로 이루어진 정방향 프라이머 및 서열번호 5에 나타낸 염기서열로 이루어진 역방향 프라이머를 이용하여 cDNA를 합성하는 단계와;
상기 cDNA를 주형으로 PCR 증폭하는 단계;를 포함하는 것을 특징으로 하는 유전자 p-psbB를 이용한 산삼의 감별방법.The method according to claim 2,
RT-PCR of the sample may include separating total RNA from ginseng or wild ginseng which is a sample;
Synthesizing cDNA using a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 4 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 5;
PCR amplification of the cDNA as a template; a method for discriminating wild ginseng using p-psbB gene, characterized in that it comprises a.
상기 RT-PCR 생성물의 확인은 2%의 아가로스겔에서 전기영동하는 방법이나, RT-PCR 생성물을 60℃에서 90℃로 0.5℃씩 온도를 증가시켜 측정하는 용융커브 분석법을 통해 이루어지는 것을 특징으로 하는 유전자 p-psbB를 이용한 산삼의 감별방법.The method according to claim 3,
Identification of the RT-PCR product is carried out by electrophoresis on 2% agarose gel, or through a melt curve analysis method for measuring the RT-PCR product by increasing the temperature by 0.5 ° C from 60 ° C to 90 ° C. Differentiation method of wild ginseng using the gene p-psbB.
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