The studies on lac operon structure genes and their flanking genes of Erwinia chrysanthemi

• Main Authors: Huang Ling-Yi, 黃令宜
• Other Authors: Lee Yung-An
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/05264305458691630595

碩士 === 輔仁大學 === 生命科學系碩士班 === 97 === Bacterial soft rot pathogen of plant is mainly caused by Erwinia chrysanthemi (Ech) and Erwinia carotovora subsp. carotovora (Ecc). These bacteria were grown in MacConkey medium and Luria medium with different colors of colonies. Luria medium contains lactose and bromothymol blue. Ech strains can’t produce organic acid with lactose, but Ecc can. Accordingly, we have focused on the structural gene types of lac operon of Ecc and Ech. Furthermore, we have investigated the flanking genes of Ech lac operon. The lacZ and lacY gene of Erwinia carotovora subsp. atroseptica SCRI1043 (Eca) could be used to detect Ech and Ecc by PCR amplification and southern hybridization. The result indicated that Ech only possess lacZ gene, but Ecc possess lacZ and lacY gene. We examined the flanking genes of lac operon in different E. coli strains, they had the same genes but differ to Eca’s lac operon flanking genes. When we used the lac operon flanking genes of Eca to detect Ech and Ecc by PCR amplification, it showed that Ecc and Eca had same genes, but Ech didn’t. Therefore, we suggested that Eca and Ech had differnt flanking genes of lac operon. We constructed lacZ gene by E. chrysanthemi Dd1200 genomic library. And found that Ech only possess lacZ and the flanking genes were aspartate racemase gene-lacI-lacZ- tRNAAsn-integrase gene after subcloning,sequencing,and sequence analysing.. Piled up and analyzed lacZ gene of Ech and Ecc could be used to design specific primers of Ech and Ecc. By combining the specific primers of lacZ and lacY, we could distinguish and characterize Ecc and Ech. Besides, we designed an intragene primer : lacZ-integrase, and tested Ech of 9 biovar by PCR amplification, four of them (biovar1,6,7,9) had correct fragment. Futhermore, we cloned lacZ gene by E. chrysanthemi PB1 genomic library. After subcloning, sequencing, sequence analysing, we found that Ech PB1 only possess lacZ, and the order of flanking genes were aspartate racemase gene-lacI-lacZ-tRNAAsn-amino acid ABC transporter gene. To sum up, the flanking gene of Ech lac operon had two different types. One was integrase gene next to lacZ gene, and another was tRNAAsn-amino acid ABC transporter gene next to lacZ gene. After analyzing the flanking genes of four bacteria’s lac operon, E. coli, Eca, Ech Dd1200 and Ech PB1, we suggested that bacteria got lacZ gene by horizontal gene transfer. The lac operon located on different sites of these bacteria. It is possible that lacZ operon was evolved from a single lacZ gene to lacZ-lacY or lacZ-lacY-lacA.