• 한국미생물·생명공학회지
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• 제16권4호
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• pp.316-319
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• 1988

토양으로부터 분리한 알카리내성 Bacillus sp. YA-14의 Pectate lyase(PL) 유전자를 E. coli에 cloning하여 제조한 재조합 plasmid pYPC29는 삽입 된 1.6kb 단편내에 PL 유전자를 함유하고 있었으며, 이 외래 DNA가 Bacillus sp. YA-14의 chromosomal DNA에서 유래된 것임을 Southern hybridization을 통하여 확인하였다. 재조합 plasmid pYPC29는 E. coli내에서 안정하게 존재하였으며 이를 함유한 재조합체의 전체 PL 활성 중 약 70%가 periplasmic space에 존재하였다.

• 한국미생물·생명공학회지
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• 제25권6호
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• pp.571-579
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• 1997

For the overproduction of pectate lyase (PL), the recombinant plasmid pl2BS fl which has strong promoter from alkali-tolerent Bacillus sp. YA-14 was used. In order to overexpress the pectate lyase by the action of overlapping strong promoter in pl2BS$\Delta$fl, 1.6 kb of PL gene was inserted into pl2BS$\Delta$fl to form pl2BS$\delta$f1-PL and the enzyme was expressed. But decreased expression efficiency of the PL gene was observed and it was due to the presence of the transcription terminator region on the upstream of the PL gene. The transcription terminator of the PL gene in pl2BS$\delta$f1-PL was removed and the resulting plasmid p12BS$\Delta$fl$\Delta$PL was formed. Bacillus subtilis 207-25 harboring the recombinant plasmid, p12BS$\Delta$fl$\Delta$PL, revealed increased expression efficiency with chloramphenicol induction when cat-86 was used as a reporter gene.

• 한국환경농학회지
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• 제40권3호
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• pp.179-185
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• 2021

BACKGROUND: Tomato genome editing using CRISPR-Cas9 is being actively conducted in recent days, and lots of plant researches have been aiming to develop high valued crops by editing target genes without inserting foreign genes. Many researchers have been involved in the manipulation of the crop ripening process because fruit ripening is an important fruit phenotype for increasing fruit shelf life, taste, and texture of crops. This paper intends to evaluate target sgRNA to edit the two ripening-related genes encoding pectate lyase (PL) and phytoene synthase (Psy) with the CRISPR-Cas9 system. METHODS AND RESULTS: The CRISPR-Cas9 expression vector was cloned to target the PL (Solyc03g111690), Psy1 (Solyc03g031860), and Psy2 (Solyc02g081330) genes, which are the ripening genes of tomatoes. Tomatoes injected with Agrobacterium containing the CRISPR-Cas9 expression vector were further cultured for 5 days and used to check gene editing efficiency. As a result of the target gene sequence analysis by the next generation sequencing method, gene editing efficiency was calculated, and the efficient target location was selected for the PL and Psy genes. CONCLUSION: Therefore, this study was aimed to establish target sgRNA data that could have higher efficiency of the CRISPR-Cas9 system to obtain the delayed ripening phenotype of tomato. The developed method and sgRNA information is expected to be utilized in the development of various crops to manage its ripening processes.

• Applied Biological Chemistry
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• 제40권5호
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• pp.380-387
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• 1997

Pectate lyase isoenzymes을 분비하는 Erwinia carotovorn subsp. carotovora LY34는 식물조직을 연화시키는 연부균이다. 이 균주로부터 게놈 DNA를 분리하여 Sau3Al 제한효소로 부분 절단한 다음 pBluescript $SK^+$ 벡터에 클로닝하여 pectate Iyase를 분비하는 클론을 분리하였다 분리 결과 4.2 kb크기의 DNA 단편을 가지고 있었으며 이를 다시 재클로닝하여 3.1 kb크기의 pelCI유전자를 함유하는 pLYPA100을 구하였다. 이 유전자의 DNA 염기서열을 분석한 결과 374 개의 아미노산을 구성하는 1,122 bp의 ORF를 확인하였다. 시작코돈과 종결코돈은 ATG와 TAA였으며 초기 서열 22개의 아미노산으로 구성된 전형적인 원핵세포의 signal peptide가 존재하였다. PeICI의 단백질 염기서열을 다른 단백질과 유사성을 분석한 결과 Erwinia carotovera subsp. carotovora Er 균주의 PelIII, Erwinia carotevora subsp. carotovora SCR193 균주의 PeIC 및 Erwinia caretovora subsp. atroseptica C18 균주의 Pel3과 유사하였으며 PLbc family에 속하였다. PeICI의 분자량은 40,507, pI는 7.60으로 계산되었다.