KR101660383B1 - Low temperature resistance transgenic gourd and method of producing thereof - Google Patents
Low temperature resistance transgenic gourd and method of producing thereof Download PDFInfo
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Abstract
본 발명은 서열번호 1의 염기서열로 구성된 애기장대 유래의 저온저항성 증진 유전자를 포함하는 재조합 벡터를 박 대목 식물 세포에 형질 전환시키는 단계를 포함하는 박 대목 형질 전환체 제조방법과 이를 이용한 저온저항성 형질 전환 박 대목에 관한 것이다.The present invention relates to a method for producing a pine needle plant transformant comprising the step of transforming a recombinant vector containing a low temperature resistance promoting gene derived from Arabidopsis thaliana comprising the nucleotide sequence of SEQ ID NO: 1 into a pine needle plant cell and a low temperature resistant strain This is related to the conversion pathway.
Description
본 발명은 박 대목를 형질전환 시킨 형질전환체에 관한 것으로, 더욱 상세하게는 저온저항성 성질을 가지는 박 대목에 대한 형질전환체 및 이의 제조 방법에 관한 것이다.More particularly, the present invention relates to a transformant having a low-temperature resistant property and a method for producing the transformant.
우리나라를 포함한 극동아시아 지역에 비해 박과 채소의 접목재배가 확대되지 않은 외국에서는 접목재배 자체에 대한 연구만이 집중적으로 수행되고 있고 대목류의 생명공학적 개량에 관한 연구는 초보적 단계에 머무르고 있다. 박과 채소의 대목 중 박의 형질전환을 통한 개량에 관한 연구는 우리나라가 독보적인 수준을 유지하고 있는 것으로 판단되는데, 우리나라에서는 경북대학교 연구진을 비롯하여 국립원예특작과학원 및 농우바이오(주) 등에서 수행하고 있다. 그러나 내건성과 내한성 등을 발현하는 목표유전자의 동정에 관해서는 선진 각국이 더 활발하게 수행하고 있다고 판단된다. 특히 내건성 형질전환 작물(옥수수, 밀 등)의 경우는 Monsanto 등의 다국적 기업에서 이미 개발 완료하여 시판을 하고 있으나 우리나라에서는 외국에서 동정된 유전자 등에 관한 재산권, LMO 안전성 평가의 경험 미흡, 법규의 엄정성 및 수용에 관한 사회적 보수성 등으로 하여 LM작물의 실용화가 지지부진한 상황이다. 이러한 상황을 고려하면 한편으로는 상대적으로 미흡한 부분을 보완하는 연구를 수행하고 다른 한편으로는 본 연구에서와 같이 우리의 강점인 형질전환 작물의 개발연구를 활발히 진행할 필요가 있다고 판단된다. In Korea, where grafting of pork and vegetables is not widespread compared to Far East Asia including Korea, research on grafting itself is concentrated, and research on biotechnology improvement of grafts remains at an early stage. In Korea, researchers at Kyungpook National University, National Horticultural Research Center, and Nongwoo Bio Co., Ltd. have conducted studies on the improvement of transgenic plants through the transformation of pork and vegetables. have. However, advanced countries are more actively involved in identification of target genes that express mild and cold tolerance. However, in Korea, there is a lack of experience in the evaluation of the property rights on genes identified in foreign countries, the experience of safety evaluation of LMOs, the strictness of laws, And social conservatism about the acceptance of LM crops. Considering this situation, it is necessary to carry out studies that complement the relatively insufficient part, and on the other hand, it is necessary to actively study the development of transgenic crops, which is our strength, as in this study.
본 발명은 애기장대 유래 저온저항성 증진 유전자를 박 대목에 도입하여 다른 특성의 변형없이 저온저항성 박 대목 형질 전환체를 개발하고자 한다.The present invention intends to develop a low-temperature-resistant, thin-walled transformant without introducing other characteristics into the low-temperature resistance promoting gene derived from Arabidopsis thaliana.
본 발명의 일 측면은 서열번호 1의 아미노산 서열로 구성된 애기장대 유래의 CBF3 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 재조합 벡터를 박 대목 식물 세포에 형질 전환시키는 단계를 포함하는 박 대목 형질 전환체 제조방법을 제공할 수 있다.One aspect of the present invention is a method for producing a transgenic plant transformant comprising the step of transforming a recombinant vector comprising a polynucleotide encoding a CBF3 protein derived from Arabidopsis thaliana comprising the amino acid sequence of SEQ ID NO: Can be provided.
또한, 상기 형질 전환은 상기 벡터를 포함하는 아그로박테리움을 박 대목 박 대목 체세포에 접종하여 수행되는 것인 제조방법을 제공할 수 있다.In addition, the transformation can be carried out by inoculating Agrobacterium containing the vector into a somatic embryo.
또한, 상기 서열번호 1의 CBF3 단백질을 코딩하는 폴리뉴클레오티드는 서열번호 2 또는 서열번호 3의 폴리뉴클레오티드인 제조방법을 제공할 수 있다.In addition, the polynucleotide encoding the CBF3 protein of SEQ ID NO: 1 may be a polynucleotide of SEQ ID NO: 2 or SEQ ID NO: 3.
또한, 상기 박 대목 형질 전환체는 저온저항성을 나타낼 수 있는 것인 제조방법을 제공할 수 있다.In addition, it is possible to provide a production method wherein the pine needle transformant can exhibit low temperature resistance.
또한, 본 발명의 일 측면은 서열번호 1의 아미노산 서열로 구성된 애기장대 유래의 CBF3 단백질을 코딩하는 폴리뉴클레오티드 또는 이를 포함하는 발현벡터가 도입되어 저온저항성을 가지는 박 대목 형질전환체를 제공할 수 있다.Another aspect of the present invention is to provide a polynucleotide encoding a CBF3 protein derived from Arabidopsis thaliana comprising the amino acid sequence of SEQ ID NO: 1 or a polynucleotide having a low temperature resistance by introducing an expression vector containing the polynucleotide .
또한, 상기 발현벡터는 도 1의 개열지도를 가지는 것인 형질 전환용 벡터를 제공할 수 있다.In addition, the above expression vector can provide a transformation vector having the cleavage map of Fig.
본 발명의 일 측면에 따른 저온저항성 박 대목 형질전환체는 저온저항성 박 대목 춤종 개발을 위한 재료로서 활용될 수 있다.The low temperature resistant strain of the present invention can be utilized as a material for the development of a low temperature tolerant bamboo root dancer.
도 1은 저온저항성을 증진시키기 위하여 삽입시킨 pBm P35sGFPHYR벡터를 나타낸 모식도이다.
도 2는 PCR을 통하여 형질전환체를 확인한 것을 나타낸 것이다.
도 3은 형질전환체의 southern 분석을 한 것이다.
도 4는 저온처리 직후 박의 모습을 나타낸 것이다.
도 5는 박의 저온처리 한 것과 저온처리하지 않은 처리 구의 파종 후50일째 생육모습을 나타낸 것이다. 각각 저온처리 구(A: 형질전환체, B: 비 형질전환체), 저온처리 하지 않은 처리구(C: 형질전환체, D: 비 형질전환체)이다.
도 6은 박의 저온저항성 검정모습을 나타낸 것이다.
도 7은 형질전환체 Nouthern 분석(Lane 1~16 형질전환체)을 한 것이다. FIG. 1 is a schematic diagram showing a pBm P35sGFPHYR vector inserted to improve low-temperature resistance.
Fig. 2 shows that the transformant was confirmed through PCR.
Figure 3 shows southern analysis of transformants.
Fig. 4 shows a state of foil immediately after the low temperature treatment.
Fig. 5 shows the appearance of growth on the 50th day after sowing of the low-temperature-treated and low-temperature-treated plants. (A: transformant, B: non-transformant), and the treatment not subjected to low temperature treatment (C: transformant, D: non-transformant).
FIG. 6 shows the low temperature resistance test of the foil.
Figure 7 shows the transformant Nouthern analysis (Lane 1-16 transformants).
본 발명의 일 측면은 국립원예특작과학원에 개발한 G5 순계계통에 CBF3 유전자가 도입된 pBm P35sGFPHYR벡터를 agrobacterium 방법을 이용하여 도입하였다. Southern분석을 실시하였는데, 형질전환체는 9개에서부터 1개까지 다양하게 유전자가 도입된 것을 확인할 수 있었다.One aspect of the present invention is a vector with a pBm P35sGFPHYR CBF3 gene introduced into the G5 sungye system developed at the National Academy of Sciences horticulture teukjak was introduced using the agrobacterium method. Southern analysis was carried out, and it was confirmed that genes were transfected from 9 to 1 in various transformants.
또한, 형질전환체의 저온저항성 실헙조건 8에서 5일간 처리하였을 때 저온에 식물체가 많은 영향을 받는 것을 확인하였다. 그리고 형질전환체의 저온저항성 검정을 위하여 CBF3 유전자가 도입된 형질전환체와 비 형질전환체를 5주를 8에서 5일간 저온처리하고 관찰한 결과 형질전환체는 저온처리 직 후 비형질전환체가 거의 고사한데 반해 살아있었다.In addition, when the transformant was treated for 5 days at a low temperature resistant condition of 8, it was confirmed that plants were affected by low temperatures. In order to test the low temperature resistance of the transformants, CBF3 gene transfected and non - transfected transformants were subjected to low temperature treatment for 5 to 8 days for 8 to 5 days. As a result, I was alive while I was dead.
파종 후 59일째 생육상황을 살펴보면 저온처리 구에서 식물체 크기가 109.7cm, 비 형질전환체는 3,8cm였고 저온처리하지 않은 처리구에서는 형질전환체가 159.8cm, 비 형질전환체가 160.6cm로 형질전환체는 정상적인 생육을 보여 저온에 저항성을 가지고 있음을 보여주고 있다.On the 59th day after sowing, the plant size was 109.7cm in the low temperature treatment and the non-transformant was 3,8cm. In the low temperature treatment, the transformant was 159.8cm and the non-transformant was 160.6cm. It shows normal growth and resistance to low temperatures.
이하 본 발명에 대해서 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
형질전환 및 식물체 재분화Transformation and plant regeneration
형질전화에 사용된 CBF3유전자는 C-repeat-binding factor (CBF3)/dehydration-responsiveelement-binding factor (DREB1A)로 애기장대로부터 유래한 유전자로 저온저항성 유전자로 알려져 있다.The CBF3 gene used in the transfection was derived from Arabidopsis as a C-repeat-binding factor (CBF3) / dehydration-responsiveelement-binding factor (DREB1A).
박 형질전환을 위해 사용한 방법은 agrobacterium을 이용한 형질전환 방법을 사용하였다. 35S promoter와 35S terminator에 의해 발현이 조절되는 CBF3 유전자와 hygromycin 저항성 유전자를 가진 pBm P35sGFPHYR 벡터를 제작하여 agrobacterium EHA101 strain에 형질전환하였다. 이 균주를 rifampicin 50 mg/L, spectinomycin 50mg/L이 포함된 LB 액체배지 3 ml에 전배양 한다. 하루 동안 28에서 균을 키운 후, rifampicin 50 mg/L, spectinomycin 50mg/L이 들어간 LB 액체배지 100ml에 배양하여 O.D.값이 0.7이 될 때까지 키운다. 형질전환을 하기 위한 박 종자는 70% 에탄올과 1% 락스를 이용하여 잘 소독한 후, 종자를 파종배지 (MS 4.4g/L, sucrose 30g/L, plant agar 8g/L, pH 5.8) 에 치상한다. 파종배지에 치상하여 5일이 지난 후, 준비한 균과 함께 공동배양을 시작한다. 잘 자란 유근을 페트리디쉬에 꺼내 놓고 자엽의 상단부 2/5 정도와 유근 부위를 자른 후 둘로 쪼갠 후 표피를 벗긴다. 표피를 벗긴 절편체를 준비된 균과 함께 교반한다. 이렇게 접종된 절편체를 공동배양 배지(MS 4.4g/L, sucrose 30g/L, MES 0.5g/L, BA 3mg/L, plant agar 8g/L, acetosyringone 50uM, pH5.2) 에 치상하여 7일이 지난 후 선발배지 (MS 4.4g/L, sucrose 30g/L, MES 0.5g/L, BA 3mg/L, plant agar 8g/L, AgNO3 0.5mg/L, cefotaxime 500mg/L, hygromycin 10mg/L, pH5.8) 에 계대배양한다. 공동배양 후 한달 정도가 지난 다음 캘러스에서 shoot가 생성되면 재분화 배지 (MS 4.4g/L, sucrose 30g/L, plant agar 8g/L, cefotaxime 500mg/L, IAA 0.1mg/L, pH5.8) 에 옮겨 뿌리가 자라도록 한다. 이렇게 해서 뿌리가 자란 식물체는 다시 병 배지 (MS 4.4g/L, sucrose 30g/L, GELRITE 8g/L)에 옮겼다.The transformation method using agrobacterium was used for the method of transforming the cells . To produce a vector with a pBm P35sGFPHYR CBF3 gene and the hygromycin resistance gene whose expression is controlled by the 35S promoter and 35S terminator was transformed in agrobacterium strain EHA101. This strain is pre-cultured in 3 ml of LB liquid medium containing 50 mg / L of rifampicin and 50 mg / L of spectinomycin. After cultivating the bacteria at 28 for one day, cultivate in 100 ml of LB liquid medium containing 50 mg / L of rifampicin and 50 mg / L of spectinomycin and cultivate until the OD value reaches 0.7. The seeds were sterilized by using 70% ethanol and 1% lactose. The seeds were seeded in a seeding medium (MS 4.4 g / L, sucrose 30 g / L, plant agar 8 g / L, pH 5.8) do. After 5 days from the date of seeding on the seeding medium, co-culture with the prepared bacteria is started. Remove the well-grown roots in a Petri dish and cut about 2/5 of the upper part of the cotyledons and cut off the roots, then split them into two and peel off the epidermis. The epidermal strips are agitated with the prepared bacteria. The seeds thus inoculated were placed in a co-culture medium (MS 4.4g / L, sucrose 30g / L, MES 0.5g / L, BA 3mg / L, plant agar 8g / L, acetosyringone 50uM, pH 5.2) L of agar, 0.5 mg / L of AgNO3, 500 mg / L of cefotaxime, 10 mg / L of hygromycin, 3 mg / L of MES, 3 mg / pH 5.8). After a month of co-cultivation, shoots were formed in the callus and then seeded in the regeneration medium (MS 4.4 g / L, sucrose 30 g / L, plant agar 8 g / L, cefotaxime 500 mg / L, IAA 0.1 mg / Let the roots grow. The plants thus grown were transferred to bottle medium (MS 4.4 g / L, sucrose 30 g / L, GELRITE 8 g / L).
DNA 추출 및 PCR 분석DNA extraction and PCR analysis
식물체가 형질전환체임을 확인하기 위하여 genomic DNA를 분리하여 PCR을 통해 확인하였다. 형질전환식물체의 잎 조직 100 mg을 액체질소를 이용하여 곱게 간 후 CTAB buffer를 이용하여 genomic DNA를 분리하였다. 이렇게 추출한 genomic DNA를 주형으로 하여 PCR을 수행하였다. 2X TB premix (Inclone)를 사용하였으며 CBF3 specific forward primer (5`- TGT TTG GCT CCG ATT ACG AG -3`), CBF3 specific reverse primer (5`- AAA AGC ATC CCT TCT GCC AT -3`)를 사용하였다. PCR 수행조건은 94에서 7분, 94에서 30초, 58에서 45초, 72에서 45초로 40주기를 수행한 후, 추가적으로 72에서 15분 동안 반응시켜준 후 종결하였다.Genomic DNA was isolated and confirmed by PCR to confirm that the plant is a transformant. 100 mg of the leaf tissue of transgenic plants was finely ground with liquid nitrogen and genomic DNA was isolated using CTAB buffer. PCR was carried out using the extracted genomic DNA as a template. 2X TB premix (Inclone), CBF3 specific forward primer (5'- TGT TTG GCT CCG ATT ACG AG -3 ') and CBF3 specific reverse primer (5'- AAA AGC ATC CCT TCT GCC AT -3`) Respectively. PCR was performed at 94 for 7 min, 94 to 30 sec, 58 to 45 sec, 72 to 45 sec for 40 cycles, followed by 72 to 15 min for reaction.
Sourthern 분석Sourthern Analysis
각 샘플당 40ug의 DNA를 사용하였고, 100units의 EcoRI으로 자른 후 DNA를 1% agarose gel에 0.5X TBE 용액에 30V로 17시간 running하였다. DNA는 nylon HybondTM-N+ membrane(Amersham Life science, UK)에 capillary transfer 방법으로 블롯팅하였다. 30ng의 CBF3 유전자로부터 유래한 PCR 산물 DNA는 RedyprimeIITM Random Prime labeling System(Amersham Life science, UK)을 사용하여 labeling 하였다. Hybridization과 blot 세척은 표준화된 방법에 따랐다(Sambrook et al. 1989). 표지인식을 위해서는 BAS-1800II Bio-Imaging analyzer(FUJIFILM)을 사용하였다.40 ug of DNA per sample was used and 100 units of EcoRI were cut and the DNA was run on 1% agarose gel in 0.5X TBE solution at 30V for 17 hours. DNA was blotted onto a nylon HybondTM-N + membrane (Amersham Life science, UK) by capillary transfer method. 30 ng of PCR product DNA derived from CBF3 gene was labeled using RedyprimeIITM Random Prime labeling System (Amersham Life science, UK). Hybridization and blot washing were in accordance with standardized methods (Sambrook et al. 1989). BAS-1800II Bio-Imaging analyzer (FUJIFILM) was used for label recognition.
Nourthern 분석Nourthern Analysis
RNA 추출은 Trizol(Invitrogen)을 이용하여 박 잎으로부터 추출하였다. RNA농도는 NanoVue(GE healthcare)로 측정하였고, 각 샘플당 30ug의 RNA를 1X MOPS buffer와 6.3%(w/v) formaldehyde가 포함된 1%(w/v) agarose gel에서 100V로 40분 간 전개시켰다. RNA는 nylon HybondTM-N+ membrane(Amersham Life science, UK)에 capillary transfer 방법으로 블롯팅하였다. 이후 실험 방법은 서던 분석 방법과 동일하다.RNA extraction was performed using Trizol (Invitrogen). RNA concentration was measured by NanoVue (GE healthcare) and 30 ug of RNA per sample was developed in 1% (w / v) agarose gel containing 1X MOPS buffer and 6.3% (w / v) formaldehyde for 40 min at 100V . RNA was blotted onto a nylon HybondTM-N + membrane (Amersham Life science, UK) by capillary transfer method. The experimental method is the same as the Southern analysis method.
저온저항성 검정Low temperature resistance test
저온저항성 검정조건을 확립하기 위하여 박대목을 본엽이 1매가 되었을때 12시간 일장, 3, 000Lux 광조건, 8에서 1일부터 7일간 처리하였다.In order to establish the low temperature resistance test condition, we treated for 12 days, 3,000Lux light condition, 8 days from 1 day to 7 days when the leaf was one leaf.
형질전환체의 저온저항성 검정을 위하여 CBF3 유전자가 도입된 형질전환체와 비 형질전환체를 5주를 8에서 5일간 저온처리하고 격리온실에 재배하고 형질전환체와 비 형질전환체 5 주를 저온처리하지 않고 격리온실에 재배한 후 생육상황을 관찰하였다.In order to test the low temperature resistance of transformants, CBF3 gene transfected and non - transfected transformants were subjected to low temperature treatment for 8 to 5 days, cultivated in an isolated greenhouse, and 5 transformants and non - After growing in isolated greenhouse without treatment, growth condition was observed.
형질전환을 통한 CBF3유전자 도입Introduction of CBF3 gene through transformation
국립원예특작과학원에 개발한 G5 순계계통에 CBF3 유전자가 도입된 pBm P35sGFPHYR벡터를 사용하여 agrobacterium 방법을 이용하여 도입하였다. 형질전환체의 확인을 위해서 기내 식물체를 CBF3 프라이머를 사용하여 PCR을 통해 먼저 선발하였다. 이를 도 1에 나타내었다. 선발된 식물체는 순화과정을 거쳐서 격리온실에서 재배하였으며, 유전자 도입 copy 수를 확인하기 위하여 southern분석을 실시하였는데, 형질전환체는 9개에서부터 1개 까지 다양하게 유전자가 도입된 것을 확인할 수 있었다. 이를 도 3에 나타내었다.Sungye on the G5 system developed at the National Horticultural Research Institute teukjak using CBF3 pBm P35sGFPHYR gene vector was introduced was introduced using the agrobacterium method. For identification of the transgenic plants, in vitro plants were first selected by PCR using CBF3 primers. This is shown in FIG. The selected plants were cultivated in isolation greenhouse through purification process and southern analysis was carried out to confirm the number of transgenic copies. It was confirmed that the transgenic plants were transfected with various genes from 9 to 1. This is shown in FIG.
형질전환체의 저온저항성 정도를 조사하기 위하여 먼저 저온저항성 조건을 ?O기 위한 실험을 실시하였는데, 8에서 실험한 결과 5일간 처리하였을 때 저온에 식물체가 많은 영향을 받는 것을 확인하였다. 이를 도 6에 나타내었다.In order to investigate the degree of low temperature resistance of the transformant, experiments were conducted to determine the low temperature resistance condition. The experiment was conducted in 8, and it was confirmed that the plant was affected at low temperature for 5 days. This is shown in Fig.
형질전환체의 저온저항성 검정을 위하여 CBF3 유전자가 도입된 형질전환체와 비 형질전환체를 5 주를 8에서 5일간 저온처리하고 관찰한 결과 형질전환체는 저온처리 직후 비 형질전환체가 거의 고사한데 반해 살아있었다. 이를 도 4를 통해 확인할 수 있다.In order to test the low temperature resistance of the transformants, the transformants transformed with the CBF3 gene and the non - transformants were subjected to low temperature treatment for 5 to 8 days. As a result, It was alive. This can be confirmed from FIG.
하기 표 1에서 보듯이 파종 후 59일째 생육상황을 살펴보면 저온처리 구에서 식물체 크기가 109.7cm, 비 형질전환체는 3,8cm였고 저온처리하지 않은 처리구에서는 형질전환체가 159.8cm, 비 형질전환체가 160.6cm로 형질전환체는 정상적인 생육을 보여 저온에 저항성을 가지고 있음을 보여주고 있다.As shown in the following Table 1, the growth rate on the 59th day after sowing was as follows: the plant size was 109.7 cm in the low temperature treatment plant and 3,8 cm in the non-transformant; in the untreated treatment, the transformant was 159.8 cm; cm, indicating that the transformant is resistant to low temperatures due to normal growth.
<110> Republic of Korea <120> Low temperature resistance transgenic gourd and method of producing thereof <130> 14-0158 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CBF3 protein <400> 1 Met Ala Ser Pro Thr Ala Pro Ser Gly Met Pro Gly Ser Ala Thr Gly 1 5 10 15 Ser Ser Val Ser Ser Gly Gly Ala Thr Ile Pro Thr Leu Ala Ser Ser 20 25 30 Cys Pro Leu Leu Pro Ala Gly Ala Leu Leu Pro Ala Gly Thr Ala His 35 40 45 Pro Ile Thr Ala Gly Val Ala Ala Ala Ala Ser Gly Leu Thr Val Cys 50 55 60 Gly Val Ala Gly Pro Ala Leu Leu Thr Ala Ile Thr Leu Gly Thr Pro 65 70 75 80 Gly Thr Ala Gly Met Ala Ala Ala Ala His Ala Val Ala Ala Leu Ala 85 90 95 Leu Ala Gly Ala Ser Ala Cys Leu Ala Pro Ala Ala Ser Ala Thr Ala 100 105 110 Leu Ala Ile Pro Gly Ser Thr Cys Ala Leu Ala Ile Gly Leu Ala Val 115 120 125 Ala Gly Ala Ala Leu Ala Pro Gly Ala Gly Met Cys Ala Ala Thr Thr 130 135 140 Ala His Gly Pro Ala Met Gly Gly Thr Leu Val Gly Ala Ile Thr Thr 145 150 155 160 Ala Gly Gly Ser Gly Ala Ala Pro Thr Met His Ala Gly Ala Met Pro 165 170 175 Gly Met Pro Ser Leu Leu Ala Ala Met Ala Gly Gly Met Leu Leu Pro 180 185 190 Leu Pro Ser Val Gly Thr Ala His Ala His Gly Val Ala Gly Ala Ala 195 200 205 Ala Ala Val Ser Leu Thr Ser Thr 210 215 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CBF3 forward <400> 2 tgtttggctc cgattacgag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CBF3 reverse <400> 3 aaaagcatcc cttctgccat 20 <110> Republic of Korea <120> Low temperature resistance transgenic gourd and method of producing it <130> 14-0158 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> CBF3 protein <400> 1 Met Ala Ser Pro Thr Ala Pro Ser Gly Met Pro Gly Ser Ala Thr Gly 1 5 10 15 Ser Ser Val Ser Ser Gly Gly Ala Thr Ile Pro Thr Leu Ser Ser Ser 20 25 30 Cys Pro Leu Leu Pro Ala Gly Ala Leu Leu Pro Ala Gly Thr Ala His 35 40 45 Pro Ile Thr Ala Gly Val Ala Ala Ala Ser Gly Leu Thr Val Cys 50 55 60 Gly Val Ala Gly Pro Ala Leu Leu Thr Ala Ile Thr Leu Gly Thr Pro 65 70 75 80 Gly Thr Ala Gly Met Ala Ala Ala Ala His Ala Val Ala Ala Lea Ala 85 90 95 Leu Ala Gly Ala Ser Ala Cys Leu Ala Pro Ala Ala Ser Ala Thr Ala 100 105 110 Leu Ala Ile Pro Gly Ser Thr Cys Ala Leu Ala Ile Gly Leu Ala Val 115 120 125 Ala Gly Ala Ala Lea Ala Pro Gly Ala Gly Met Cys Ala Ala Thr Thr 130 135 140 Ala His Gly Pro Ala Met Gly Gly Aly Ile Thr Thr 145 150 155 160 Ala Gly Gly Aly Ala Aly Gly Aly Gly Aly Met Pro 165 170 175 Gly Met Pro Ser Leu Leu Ala Ala Met Ala Gly Gly Met Leu Leu Pro 180 185 190 Leu Pro Ser Val Gly Thr Ala His Ala His Gly Val Ala Gly Ala Ala 195 200 205 Ala Ala Val Ser Leu Thr Ser Thr 210 215 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CBF3 forward <400> 2 tgtttggctc cgattacgag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CBF3 reverse <400> 3 aaaagcatcc cttctgccat 20
Claims (6)
상기 형질 전환은 상기 벡터를 포함하는 아그로박테리움을 박 대목 체세포배에 접종하여 수행되는 것인 제조방법.The method according to claim 1,
Wherein the transformation is carried out by inoculating an Agrobacterium containing the vector into a somatic somatic embryo.
상기 서열번호 1의 CBF3 단백질을 코딩하는 폴리뉴클레오티드는 서열번호 2의 CBF3 specific forward primer 또는 서열번호 3의 CBF3 specific reverse primer를 포함하는 폴리뉴클레오티드인 제조방법.The method according to claim 1,
Wherein the polynucleotide encoding the CBF3 protein of SEQ ID NO: 1 is a polynucleotide comprising a CBF3 specific forward primer of SEQ ID NO: 2 or a CBF3 specific reverse primer of SEQ ID NO: 3.
상기 발현벡터는 도 1의 개열지도를 가지는 것인 박 대목 형질전환체.6. The method of claim 5,
Wherein the expression vector has a cleavage map of FIG. 1.
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| KR100933320B1 (en) | 2007-11-05 | 2009-12-22 | 주식회사 에프앤피 | Transgenic Plant with Increased Resistance to Various Abiotic Stresses Transduced with AtBG1 Gene |
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| KR100933320B1 (en) | 2007-11-05 | 2009-12-22 | 주식회사 에프앤피 | Transgenic Plant with Increased Resistance to Various Abiotic Stresses Transduced with AtBG1 Gene |
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