| In order to analyze the translocation pathway of HpaXm in Xanthmonas citri subsp. malvacearum, in this
study, we constructed the hpaXm gene mutant and complementary vector. According to the relative fragments and the
conservative domain of complete sequences of X. citri subsp. malvacearum hpaXm gene and its homologous gene hpa1,
recorded by the GenBank, a series of primers were designed and synthesized. The gene knockout vector of hpaXm was
mobilized into X. citri subsp. malvacearum strains by electrotransformation. The ability of hpaXm mutant to elicit
hypersensitive responses in tobacco plants were tested. And the fragment of 349 bp upstream and 465 bp downstream of hpaXm gene were obtained from X. citri subsp. malvacearum by PCR. After the fragment of upstream and downstream of hpaXm gene were successfully fused by overlap extension PCR, it was cloned into pK18mob vector and transformed into E.
coli DH5琢. The transformed bacteria was resistant to kanamycin. The hpaXm mutant was obtained after the
electrotransformation and its capacity to elicit hypersensitive responses decreased greatly in tobacco leaves. The complete
sequences (hpaXm, 402 bp) and signal peptide similar sequences missed hpaXm gene (hpaXm46-402, 360 bp) were obtained
by PCR. Then the plasmid pHM1 was fused with hpaXm and hpaXm46-402. The recombinant constructs, pHM1hpaXm and
pHM1hpaXm46-402 were transferred to E. coli DH5 and the transformed bacteria was resistant to spectinomycin. Then the
plasmids were identified by PCR amplification and sequencing. The purpose of this study is to construct the hpaXm mutant
and complementary vector and laid the foundation for the construction of hpaXm gene reversal mutation and the further
studies on the function of hpaXm signal peptide similar sequences. K |
