| 王慕瑾，刘悦，孙超，缪卫国，郑服丛.棉花角斑病菌hpaXm基因突变体
与互补载体构建[J].广东农业科学,2014,41(12):75-81 |
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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 hpaXmgene in Xanthmonas citri subsp. malvacearum |
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| DOI： |
| 中文关键词: HpaXm 信号肽类似序列 突变体 互补载体 |
| 英文关键词: HpaXm signal peptide similar sequences mutant complementary vector |
| 基金项目:国家自然科学基金(31360029，31160359)；教育部
博士点基金(20124601110004，20104601110004)；国家重大基础
研究计划项目(2011CB111612)；国家农业产业技术体系建设项
目(CARS-34-GW8) ；海南大学科研启动资金( kyqd1006) |
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| 中文摘要: |
| 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 DH5琢，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“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 DH5琢，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。 |
| 英文摘要: |
| 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 |
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