대두 세포내에서 Glycinin 전구체의 존재 확인

김정호(Chung Ho Kim),최양도(Yang Do Choi) 한국식물학회 1989 Journal of Plant Biology Vol.32 No.1

Glycinin is the major storage protein in soybean. It has been known that a molccule of glycinin is composed of 6 subunits, each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one (MW 39K and 19K, respectively). To study the molccular origin and the relationship of glycinin subunit polypeptides, antibodies against A- and B-polypeptide were obtained by immunizing rabbits with either of the antigens purified by gel filtration and preparative electrophoresis. Each antibody was not only specific for its own antigen polypeptide in soybean but also recognized the precursor which was synthesized in vivo and in vitro. The polyadenylated mRNAs were isolated from immature seeds and leaves and were translated in vitro using wheat germ extract. One of the seed-specific translation products, MW 60K, was identified to be the precursor of glycinin subunit by immunoprecipitation with antibodies against glycinin A- and B-polypeptide. Mature A- and B-polypeptides were not detected in the translate in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield A- and B-polypeptide which form a glycinin subunit in the cell. Glycinin genes were expressed with the maturation of soybean seeds in a tissue-specific and developmental stage-specific manner.

Molecular Cloning of cDNA Encoding the Precursor to the Glycinin $A_2B_{1a}$ Subunit of Soybean

김정호,최양도,Kim, Chung-Ho,Choi, Yang-Do 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

Glycinin은 대두의 주된 저장 단백질이다. Glycinin 분자는 6개의 subunit으로 이루어져 있으며, 각 subunit은 산성 및 염기성 polypeptide로 구성되어 있다. 이들 polypeptide는 전구체로 합성되어진 후 내부 절단에 의해 산성 및 염기성 polypeptide로 갈라진다. Glycinin 유전자의 구조와 발현 메카니즘을 규명하기 위해 cDNA 유전자 발현 은행을 제조한 후, 항체를 사용한 면역 선별법에 의해 cDNA 유전자를 선별하였다. cDNA 유전자 발현 은행은 mRNA를 이용하여 double stranded cDNA를 합성한 후 ${\lambda}$gt11 발현 운반체에 삽입 제조하였다. 항체는 glycinin 산성 및 염기성 polypeptide 각각에 대한 것을 사용했는데, 이들의 항원은 mRNA 에서 합성된 전구체 단백질의 N-terrninal과 C-terminal 에 해당한다. 8개의 cDNA clone을 분리하였는데 4개는 산성 polypeptide에 대한 항체, 4개는 염기성 polypetide에 대한 항체에 의해 선별된 것이다. 염기서열을 결정한 결과, 그 중 cDNA 길이가 1185 bp인${\lambda}$G1A1 clone 은 glycinin $A_2B_{1a}$ subunit을 code하는 cDNA 유전자의 부분 clone임을 알았다. 이 clone은 산성 polypeptide의 절반과 염기성 polypeptide의 전부에 해당하였다. 이 cDNA 유전자에 해당하는 mRNA의 크기는 약 1.8 kb 임을 Northem blot 결과 알 수 있었다. Glycinin 유전자는 대두 종자에서만 발현이 되었으며, 이러한 조직 특이성 발현 형태는 transcription 단계에서 조절이 이루어지는 것을 알 수 있었다. Glycinin is the most abundant storage protein in soybean. A molecule of glycinin is composed of 6 subunits each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one. These polypeptides are synthesized as a precursor which is cleaved to yield A- and B-polypeptides. To study the structure and expression mechanism of the gene, isolation of cDNA clones for soybean glycinin was attempted by immunological screening of a soybean cDNA library with antibodies. The cDNA expression library was constructed with double stranded cDNA of polyadenylated mRNA into ${\lambda}$gt11 expression vector. Antibodies to A- and B-polypeptides of glycinin subunit which correspond to N- and C-terminal domain of mRNA translate, respectively, were employed. Eight clones for glycinin, 4 with anti-A and 4 with anti-B polypeptide antibody, were isolated by immunoscreening. The clone ${\lambda}$G1A1 with 1186 bp insert was characterized to be a partial cDNA clone for glycinin subunit $A_2B_{1a}$ by nucleotide sequencing analysis and the expression in E. coli. It encodes a half of $A_2$- and entire $B_{1a}$-polypeptide. The mRNA size of glycinin was demonstrated to be 1.8 kb by Northern blot analysis. It was expressed only in seeds maintaining tissue-specific expression pattern, which was controlled at the level of transcription rather than at translation.

대두 glyciin subunit A2B1 의 cDNA 유전자 분리

김정호,최양도 ( Chung Ho Kim,Yang Do Choi ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Glycinin is the most abundant storage protein in soybean. A molecule of glycinin is composed of 6 subunits each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one. These polypeptides are synthesized as a precursor which is cleaved to yield A- and B-polypeptides. To study the structure and expression mechanism of the gene, isolation of cDNA clones for soybean glycinin was attempted by immunological screening of a soybean cDNA library with antibodies. The cDNA expression library was constructed with double stranded cDNA of polyadenylated mRNA into λgt11 expression vector. Antibodies to A- and B-polypeptides of glycinin subunit which correspond to N- and C-terminal domain of mRNA translate, respectively, were employed. Eight clones for glycinin, 4 with anti-A and 4 with anti-B polypeptide antibody, were isolated by immunoscreening. The clone λG1A1 with 1186 bp insert was characterized to be a partial cDNA clone for glycinin subunit A₂B_(1a) by nucleotide sequencing analysis and the expression in E. coli. It encodes a half of A₂- and entire B_(la)-polypeptide. The mRNA size of glycinin was demonstrated to be 1.8 kb by Northern blot analysis. It was expressed only in seeds maintaining tissue-specific expression pattern, which was controlled at the level of transcription rather than at translation.

대두 원형질체와 형질전환된 담배에서의 대두 glycinin 유전자 Gy2 promoter 의 발현조절 기작

김수정(Soo Jung Kim),이지영(Jee Young Lee),김정호(Chung Ho Kim),최양도(Yang Do Choi) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.5

To study the regulatory expression mechanism of soybean glycinin gene, Gy2, the 5` upstream region of the gene was searched for the presence of putative regulatory elements by nucleotide sequencing. It revealed various kinds of regulatory sequence elements commonly found in plant storage protein genes. There were canonical promoter sequences, TATA box (TATAAT) and AGGA box (GAAT) which are common in the 5` upstream region of the plant genes. The embryo factor binding sequence, RY repeat, CACA sequences, α-conglycinin enhancer-like sequences were also found. To delineate the function of these sequences, 5` upstream deletion mutants of Gy2 were prepared and fused to the α-glucuronidase (GUS) gene. Each chimeric construct was transferred into soybean protoplasts for transient assay, which led to the identification of the sequences between -281 and - 223, -170 and -122, of Gy2 promoter as negative regulatory elements, and the sequences between -223 and -170, -122 and -16 as positive regulatory elements. These results are consistent in transformed tobacco plants as well. The serially deleted promoter fragments fused to the GUS were transformed into Nicotiana tabacum by Agrobacterium tumefaciens using the binary vector system. GUS activity of Gy2 promoter deletion constructs was detected only in seeds but not in leaves with different levels of expression as in transient assay. These results suggest that the glycinin Gy2 promoter drives a tissue-specific expression in transgenic tobacco plants.

리블로스 1,5 - 이인산 탄산화효소 유전자의 분리 및 특성 규명

박성순(Sung Soon Park),김희진(Hee Jin Kim),김정호(Chung Ho Kim),김한집(Han Jip Kim),이종섭(Jong Seob Lee),이광응(Kwang Woong Lee),최양도(Yang Do Choi) 한국응용생명화학회 1994 Applied Biological Chemistry (Appl Biol Chem) Vol.37 No.5

To study the light-induced expression mechanism and protein transport into the chloroplast, a rice genomic clone (GrbcS) for the small subunit of ribulose 1,5-bisphosphate carboxylase (rbcS) was isolated and its nucleotide sequence was determined. Nucleotide sequence analysis of GrbcS revealed that the gene consists of two exons interrupted by an intron, encoding a protein of 175 amino acids including a transit peptide of 47 amino acids. These structural features of GrbcS are consistent with those of other rbcS genes from monocot species. Genomic Southern blot analysis suggested that the rbcS genes are present as a relatively small multigene family in the rice genome. Comparison of the nucleotide and deduced amino acid sequences to other rice rbcSs shows close sequence similaritiy. Conserved DNA sequences present in other light-responsive genes are also found in the 5′ upstream region of GrbcS such as G-box, 3AF1-binding site and GATA site. The possible function of these putative regulatory elements are discussed.

담배 모자이크 바이러스와 대두 Glycinin 유전자의 5' Leader Sequence 를 이용한 외래 유전자의 전이효율 증진

강홍구(Hong Gu Kang),박지원(Jee Won Park),김정호(Chung Ho Kim),임재윤(Jae Yun Lim),최양도(Yang Do Choi) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.3

To increase the expression of a foreign protein in transgenic plant, the benefits of 5`-untranslated leader sequences of tobacco mosaic virus (TMV) RNA or soybean glycinin gene, Gy2, fused to a protein coding sequence were exploited. pGA643-derived plasmid contains 35S promoter of cauliflower mosaic virus, protein coding sequence of maize 10 kDa zero (10kZ) and Gy2 terminator. The leader from Gy2 or TMV RNA was inserted between the promoter and the coding sequence in each construct. The recombinant DNAs were introduced into tobacco plants by Agrobacterium mediated leaf disc transformation method. Although the transgene without the leader had more transcripts than the others, mRNAs containing the leader were translated more efficiently. It might be due to difference in the length of 5`-untranslated sequence and context surrounding the AUG colon, but could be sequence specific rather. These results suggest that the leader sequences of Gy2 and TMV play important roles as an enhancer in translational control of foreign gene in transgenic tobacco plant.

대두 Kunitz Trypsin Inhibitor 전구체의 동정

김정호,김수일,최양도 한국농화학회 1989 Applied Biological Chemistry (Appl Biol Chem) Vol.32 No.3

Three classes of proteinase inhibitors are known in soybean; the Kunitz trypsin inhibitor (SKTI), the Bowman-Birk proteinase inhibitor and its isoinhibitors. To study the molecular structure and expression characteristics of the SKTI, antibody was obtained by immunizing rabbit with the SKTI purified from soybean by preparative electrophoresis. Anti-SKTI antibody was not only specific for mature SKTI in soybean seed but also recognized the precursor which was synthesized in vitro. Translation in vitro was carried out in wheat Bern: extract with polyadenylated mRNA isolated from developing soybean seeds. One of the seed specific translation products, MW 24K, was identified to be the precursor for the SKTI by immunoprecipitation with anti-SKTI antibody. Mature SKTI of MW 20K, however, was not detected in the translates in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield mature SKTI in soybean seed. The SKTI gene was expressed with the maturation of soybean seed in a tissue-specific and development stage-specific manner.

대두 단백질 분해효소 억제제의 cDNA 유전자 분리

송상익,김수일,김정호,백승준,김희진,최양도,이수열 서울대학교농과대학농업개발연구소 1989 서울대농학연구지 Vol.14 No.2

A cDNA clone for the soybean trypsin inhibitor(Kunitz-type: SKTI) was isolated by immunological screening of a soybean lamda gtll expression vector cDNA library with antibody. The cDNA insert size of the clone lamda Ti-4 was 308 bp. Nucleotide sequencing of the clone shows open reading frame of 201 bp which encodes 67 amino acids of the SKTI precursor and 107 bp of 3'-nontranslating region including a part of the poly(A) tail. Comparision of deduced amino acid sequence reveals that the clone Ti-4 was partial cDNA clone of SKTI. SKTI precursor contains a stretch of 11 amino acid at C-terminal suggesting that the precursor is processed at C-terminal to produce mature SKTI in soybean seeds. Northeran blot analysis demonstrated the mRNA size of SKTI to be about 900 bp. It was expressed only in soybean seeds suggesting tissue specific gene expression was regulated at the level of transcription.