棉花角斑病菌hpaXm基因突变体与互补载体构建

Mutant and complementary vector constuction of hpaXm gene in Xanthmonas citri subsp. malvacearum

为了分析研究Harpin蛋白转运途径,针对来自棉花角斑病菌的HpaXm,构建hpaXm基因突变体和互补载体,根据文献和GenBank中已登录的hpaXm基因全序列及其同源基因hpa1的序列,设计合成多对引物进行PCR扩增,并将扩增片段与载体相连接,用电转化的方式将构建成功的hpaXm基因敲除载体转化Xanthmonas citri subsp.malvacearum,然后测定hpaXm基因突变体在烟草上激发HR的能力。结果得到hpaXm基因上游序列349 bp,hpaXm基因下游序列465 bp;利用重叠PCR将hpaXm基因上下游序列融合,克隆到自杀载体pK18mob的酶切位点BamHI和EcoRI之间,并将pK18mobhpaXm上下游融合片段转化大肠杆菌E.coli DH5α,经卡那霉素平板筛选得到阳性转化子;将hpaXm基因敲除载体转化X.citri subsp.malvacearum,经PCR验证成功敲除hpaXm基因,并发现hpaXm基因突变体在烟草上激发HR的能力要弱于野生型菌株。扩增得到hpaXm基因片段402 bp和信号肽类似序列缺失hpaXm46-402基因片段360 bp,克隆到广宿主载体pHM1的KpnI和SacI酶切位点之间,并将pHM1hpaXm和pHM1hpaXm46-402转化E.coli DH5α,经壮观霉素平板筛选得到阳性转化子。重组质粒经PCR检测、测序鉴定及突变体在烟草上的表型验证,表明hpaXm基因突变体和互补载体构建成功,为hpaXm基因回复突变株的构建和进一步研究信号肽序列缺失对HpaXm蛋白转运的影响奠定了基础。

In order to analyze the translocation pathway of HpaXm in Xanthmonas cirri 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. cirri subsp, malvacearum hpaXm gene and its homologous gene hpal, recorded by the GenBank, a series of primers were designed and synthesized. The gene knockout vector of hpaXm was mobilized into X. cirri 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. cirri 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 DH5ct. 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 priM1 was fused with hpaXm and hpaXm46-402. The recombinant constructs, pHMlhpaXm and pHMlhpaXm46-402 were transferred to E. coli DHSot 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.