Incorportation of Foreign Gene with Ti Plasmid Vector System: (I) Introduction of E. coli Thioredoxin Gene into A. tumefaciens.

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Soon-Joo,Kim, Seong-Wan,Lim, Chang-Jin,kim, Young-Myeong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

외래 유전자를 도입함으로써 식물체에 새로운 유전정보를 부여하려는 노력이 다각적으로 수행되어 왔으며 최근에는 넓은 숙주범위를 갖는 Ti plasmid를 vector로 이용한 외래 유전자의 도입이 활발히 진행되고 있다. 본 연구에서는 Ti plasmid vector의 일종인 pGA658을 이용하여 광합성 조절 등 여러가지 기능을 갖고 있는 것으로 알려져 있는 thioredoxin 유전자를 식물체내로 도입하여 식물체내에서의 그 역할을 조사할 목적으로 DNA 재조합을 시도하였다. E. coli thioredoxin 유전자를 함유하는 pCJF 4의 Hind III-BamHI DNA fragment를 pGA658의 Hind III-Bgl II site에 삽입시키고 E. coli에 transformation 한 후 형질전환체의 확인은 항생물질 marker에 대한 저항성으로, 재조합 DNA의 유전자 지도는 여러가지 제한효소를 이용한 절편의 크기 확인으로, 그리고 도입 유전자의 발현은 효소 활성 측정에 의 하여 확인하였다. 이렇게 확인된 재조합된 plasmid pKDB3를 담배세포로 도입하기 위한 전 단계로서 freeze-thaw 방법으로 Agrobacterium에 transformation 한 후 항생물질 저항성과 제한효소를 이용한 절편의 크기 확인 및 효소 활성 측정에 의하여 pKDB3가 도입되어 안정하게 유지됨을 확인할 수 있었다. In this part of study on the incorporation of foreign gene into plant cells, a derivative of Ti plasmid vector (pGA658), containing E. coli thioredoxin gene, was prepared and introduced into Agrobacterium tumefaciens. A recombinant plasmid, pKDB3, was constructed by transferring HindlII-BamHI DNA fragment of pCJF4, including E. coli thioredoxin gene, into HindIII-BglII restriction sites of plasmid pGA658. By doing this, E. coli thioredoxin gene is expected to express from nos promoter of pGA658 after the incorporation into plant cells. The structure of DNAs isolated from kanamycin-resistant E. coli transformants was convinced by restriction mapping. As a preceding step before incorporation into plant cells, the recombinant plasmid pKDB3 was transformed into A. tumefaciens by freeze-thaw procedure. In Agrobacterium transformants, the expression of E. coli thioredoxin gene was positively observed, and this suggested the stable existence of the E. coli gene.

The Incorporation of Eucaryotic Gene Using Ti Plasmid Vector: I. The Introduction of Yeast Homoserine Dehydrogenase Gene into Agrobacterium tumefaciens

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Sun-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1

효모에 있어서 threonine과 methionine 생합성에 관여하는 효소들 중 하나인 homoserine dehydrogenase가 식물체내로 도입된 후, 그 역할을 조사하고자 Ti plasmid에서 유도된 pGA658 vector와 homoserine dehydrogenase 유전자 함유 DNA의 재조합을 시도하였다. 그 결과 pGA658 vector의 nos promoter 다음에 효모의 homoserine dehydrogenase 유전자를 함유한 DNA 절편이 방향이 반대로 삽입된 재조합 plasmids가 성공적으로 만들어졌다. 형질전환된 E. coli와 Agrovbacterium tumefaciens는 적절한 항생물질 함유배지에서 저항성을 나타내었으며, 그들의 plasmid를 분리하여 여러가지 제한효소를 이용한 크기를 조사해 보니, 예상되는 크기와 일치하는 것을 관찰할 수 있었다. 또한 형질전환된 E. coli와 A. tumefaciens내에서의 yeast homoserine dehydrogenase 유전자 발현을 조사하기 위해 그 효소의 활성을 조사해 본 결과, 형질전환된 E. coli에서는 대조군에 비해 약간의 증가를 나타내었으나, 형질전환된 A. tumefaciens에서는 변화가 없었다. DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli, but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

Ti Plasmid Vector System 을 이용한 외래 유전자의 도입 : ( 1 ) A . tumefaciens 로의 E . coli Thioredonxin 유전자의 도입

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Soon Joo Hong Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

In this part of study on the incorporation of foreign gene into plant cells, a derivative of Ti plasmid vector (pGA658), containing E. coli thioredoxin gene, was prepared and introduced into Agrobacterium tumefaciens. A recombinant plasmid, pKDB3, was constructed by transferring HindIII-BamHI DNA fragment of pCJF4, including E. coli thioredoxin gene, into HindIII-BgIII restriction sites of plasmid pGA658. By doing this, E. coli thioredoxin gene is expected to express from nos promoter of pGA658 after the incorporation into plant cells. The structure of DNAs isolated from kanamycin-resistant E. coli transformants was convinced by restriction mapping. As a preceding step before incorporation into plant cells, the recombinant plasmid pKDB3 was transformed into A. tumefaciens by freeze-thaw procedure. In Agrobacterium transformants, the expression of E. coli thioredoxin gene was positively observed, and this suggested the stable existence of the E. coli gene.

Ti Plamid Vector System 을 이용한 외래 유전자의 도입 : ( 2 ) E . coli thioredoxin 유전자의 배양된 담배세포내 발현

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Soon Joo Hong,Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

This study was performed to observe the expression of E. coli thioredoxin gene incorporated in tobacco cells. The recombinant DNA used, pKDB3, had been constructed from a Ti plasmid vector pGA658 and a bacterial plasmid pCJF4 harboring E. coli thioredoxin gene, as described in the preceding paper (Lee et al., 1988). The leaf discs of plant (N. tabacum cv Xanthi) were transformed to kanamycin resistance by the cocultivation with Agrobacterium A281 containing plasmid pKDB3. Transformed leaf discs were cultured in MS agar medium with kanamycin for callus induction. Kanamycin-resistant tobacco calli were continuously grown in MS agar medium for shoot induction, and then in MS agar medium for root induction. Expression of E. coli thioredoxin gene in the plant tissue regenerated from transformed tobacco cells was confirmed by DTNB assay. The thioredoxin activity of transformed tobacco cells was much higher (about 9 times) than that of normal tobacco cells. Our results suggest that E. coli thioredoxin gene was successfully incorporated into tobacco cells, and the incorporated bacterial gene could be expressed at a high level.

Ti plasmid vector 를 이용한 진핵세포 유전자의 도입에 관한 연구

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Sun Joo Hong,Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1

DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli. but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

Incorporation of Foreign Gene with Ti Plasmid Vector System: (II) Expression of E. coli Thioredoxin Gene in Cultured Tobacco Cells.

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Soon-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

본 연구에서는 E. coli thioredoxin 유전자를 함유하고 있는 재조합된 plasmid pKDB3의 담배세포내로의 도입을 시도하고, 도입된 thioredoxin 유전자의 발현을 조사하였다. 담배(Nicotina tabacum cv Xanthi) 세포로의 재조합 plasmid pKDB3의 도입은 담배잎 절편과 재조합 DNA 및 helper Ti plasmid pTiBo 542를 함유하고 있는 Agrobacterium tumefaciens strain A281과의 cocultivation 방법을 이용하여 행하여졌으며, 형질전환된 담배세포는 항생물질 저항성 배지에서의 callus 형성 여부로 선별되었고, 선별된 형질전환된 calli는 shoot와 root 형성을 위해 적절한 MS agar 배지에서 계속 키워졌다. 이와 같이 형질전환된 담배세포에서 완전한 식물체로 재생된 담배잎에서의 E. coli thioredoxin 유전자의 발현을 조사한 결과 thioredoxin 활성이 형질전환된 담배세포가 정상세포에 비해 9배 정도 더 큰 것으로 나타났다. 이러한 일련의 결과들은 E. coli thioredoxin 유전자가 성공적으로 담배세포에 들어가서 높은 수준으로 발현됨을 보여주고 있다. This study was performed to observe the expression of E. coli thioredoxin gene incorporated in tobacco cells. The recombinant DNA used, pKDB3, had been constructed from a Ti plasmid vector pGA658 and a bacterial plasmid pCJF4 harboring E. coli thioredoxin gene, as described in the preceding paper (Lee et al., 1988). The leaf discs of plant (N. tabacum cv Xanthi) were transformed to kanamycin resistance by the cocultivation with Agrobacterium A281 containing plasmid pKDB3. Transformed leaf discs were cultured in MS agar medium with kanamycin for callus induction. Kanamycin-resistant tobacco calli were continuously grown in MS agar medium for shoot induction, and then in MS agar medium for root induction. Expression of E. coli thioredoxin gene in the plant tissue regenerated from transformed tobacco cells was confirmed by DTNB assay. The thioredoxin activity of transformed tobacco cells was much higher (about 9 times) than that of normal tobacco cells. Our results suggest that E. coli thioredoxin gene was successfully incorporated into tobacco cells, and the incorporated bacterial gene could be expressed at a high level.

Agrobacterium tumefaciens의 Ti plasmid 재조합에 관한 연구

주충노,이희봉,김영명 연세대학교 자연과학연구소 1987 學術論文集 Vol.19 No.-

Crown gall의 병원체로 알려진 Agrobacterium tumefaciens가 함유하고 있는 Tumor inducing(Ti) plasmid가 식물 hormone과 opine의 합성원인이라는 것이 밝혀지고 식물 세포의 crown gall에는 Ti plasmid가 존재하며 Ti plasmid의 일부가 식물세포의 핵 genome에 삽입되어 형질 변화가 일어남이 보고 되었고 Ti plasmid의 식물체로의 gene도입 vector로서의 연구가 최근 활발히 진행되고 있으며 현재 PLGV vector와 PMON vector 등이 개발되고 있으나 조작이 복잡하고 아직은 초보적 단계인 것으로 알려져 있다. 본 연구에서는 원하는 식물 gene을 식물체에 도입하는 유용한 vector개발의 초보단계로서 Ti plasmid의 T-DNA의 일부인 nos gene을 분리하여 pBR322와의 재조합을 기도한 것이다. pBR 322를 포함하는 E. coli로 부터 Horwitz(1979)의 방법으로 pBR 322를 분리하였고 A. tumefaciens C58에서 Kado(1979)의 방법으로 Ti plasmid를 분리한 후 Ti plasmid 내의 nos gene을 분리하였다. 분리된 pBR 322를 Hind Ⅲ로 절단한 후 CIP로 처리하고 T_4 DNA ligase를 이용하여 Hind Ⅲ로 처리한 nos gene fragment와 재조합하였다. 이와같이 하여 얻은 recombinant DNA로 인한 E. coli HB101의 형질 변화를 Mandel과 Higa의 방법에 따라 조사한 결과 pBR 322의 tetracyclin gene 사이에 nos gene이 삽입된 것으로 확인되었다. 즉 ampicillin 배지에서는 92개의 colony가 형성되었는데 그중 59개는 tetracyclin 배지에서 colony를 형성하지 못하였다. 이러한 tetracyclin 감수성이면서 ampicillin 저항성인 colony를 배양하여 얻은 recombinant plasmid를 전기이동법으로 분리한 결과 예상대로 크기가 7.5kb 정도였고 Hind Ⅲ로 절단하였을 때 2개의 band가 확인되었다. 본 연구에서 얻은 nos gene-pBR 322 hybrid plasmid는 원하는 식물 gene을 이것에 삽입하여 A. tumefaciens 내에서 homologous recombination을 행한 후 식물체에 감염시켜 원하는 gene의 발현여부를 검출하는데 이용될 것으로 기대된다. It is mow realized that the tumor-inducing(Ti) plasmid in Agrobacterium tumefaciens is responsible for the induction of crown gall tumors in dicotyledonous plants and it has been demonstrated that a segment of the Ti plasmid, so called T-DNA, is stably integrated into and expressed in the genome of transformed plant cells. Recently, the use of A. tumefaciens Ti plasmid as a vector to introduce a foreign gene has been intensively studied and several vectors such as PLGV vector and PMON vector has been reported. However, their practical use is still at a primitive stage. It was attempted in the present study, therefore, to conduct the recombination of nos gene of T-DNA from A. tumefaciens Ti plasmid and E. coli pBR 322 as a vector it introduce foreign plant gene into dicotyledon plant cells. pBR 322 was isolated from E. coli containing pBR 322 accdrding to Horowitz(1979) and nos gene of T-DNA in Ti plasmid from A. tumefaciens C58 was obtained according to Kado(1979). The isolated pBR 322 was cut using Hind Ⅲ followed by the addition of CIP·pBR 322 fragments and Hind Ⅲ treated nos gene fragments were than recombined using T_4 DNA ligase and the E. coli cells were then transformed by recombinant DNA according to Mandel and Higa(1970). It was confirmed that nos gene was inserted into tetracyclin gene of pBR 322 by the finding that when the transformed cells were incubated in the ampicillin medium, 92 colonies were formed, in which 59 were tetracyclin sensitive. The above tetracyclin sensitive but ampicillin resistant cells were then cultured and the recombinant pBR-nos gene was isolated and subjected to electrophoresis. It was found that size of the recombinant DNA was approximately 7.5 Kb as expected and two bands were appeared on electrophoretogram when the recombinant DNA was treated with Hind Ⅲ. It might be possible to insert the foreign plant genes to the pBR-nos gene hybrid plasmid obtained in the present study and the resultant DNA could be then homolgously recombined with Ti plasmid of A. tumefaciens and the final recombined Ti plasmid could be used for the introduction of the foreign plant genes into dicotyledon plant cells.