| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 82 === Up to now , only homologous plasmids , such as pLUL631 、
pLUL612 , ...etc.,were be able to be transformed into reuterin-
producing Lactobacillus reuteri;(RPLR) at high frequencies.Also
for the time being, no efficient cloning vector is avaiable for
RPLR.Under such a circumstance,identifying parts(i.e.,replicon
、 promoter and R-gene)directly from homologous plasmids of
RPLR is the best initiate way for constructing cloning vector
for RPLR. Five local RPLR strains,among them four were
resistant to erythromycin(EM) and one was resistant to
chloramphenicol(CM) , were selected for this studies. We
isolated plasmids from these antibiotic resistant strains by
CsCl-EthBr gradient centrifuga- tion method and transformated
them into RPLR type strain DSM20- 016, which is free from any
plasmid, by electroporation method. By analyzing antibiotic
resistant transformants acquired in this experiment. We were
able to identify four EM resistant plasmids(which were pTE31
【4.4 kb】、pTE32【7.0 kb】、pTE33【7. 0 kb】,and pTE36【15.0 kb
】) and one CM resistant plasmid(which was pTC88【7.0 kb】;
Further confirmation for these R-plasmids as originating from
donor strains were conducted by ① minimum inhibitory
concentration test,②restriction DNA length analysis, and ③
southern blotting hybridization . We also cloned and expressed
R-gene fragments from these five plasmids into E. coli TG1 and
E. coli DH5α by pUC19 cloning system; Five recombinant
plasmids including pUE3116(4.3 kb;containing a 1.6kb e■DNA
fragment),pUE3220(4.7kb;containing a 2.0 kb e■DNA fragment),
pUE3320(4.7 kb;containing a 2.0 kb e■ DNA fragment),
pUE3628(5.5 kb;containing a 2.8kb e■DNA fragment), and
pUC8835(6.2 kb; containing a 3.5 kb c■ DNA fragment )were
obtained in this experiment. This is the first success of
cloning and expressing c■ gene fragment from RPLR into E.
coli. These R-gene fragments, after further sequencing,will be
a good source of marker parts for constructing cloning vector.
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