CONSTRUCTION AND BIOLOGICAL CHARACTERISTICS ANALYSIS OF CPXRA MUTANT IN AEROMONAS HYDROPHILA
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摘要: 为了探讨Cpx系统在嗜水气单胞菌生长及毒力等方面发挥的作用, 利用融合PCR和基因同源重组原理, 以自杀质粒pRE112为载体构建缺失57—1879 bp序列的cpxR-A基因簇突变株 Δcpx, 然后比较缺失株和野生株在生长、生物膜形成、应激耐受及毒力等生物学特性方面的差异。普通PCR及荧光定量PCR结果验证了突变株中cpxRA基因簇片段的部分缺失, 表明突变株构建成功; 生物学特性研究结果显示, 突变株在形态、生长、生物膜形成及毒力等方面与野生株没有显著差异, 两者主要在应对高渗透压、SDS (十二烷基磺酸钠)刺激及含有EDTA (乙二胺四乙酸二钠)或多黏菌素B环境表现不同。结果表明Cpx双组分系统在嗜水气单胞菌应对外界刺激方面扮演着重要角色, 但在毒力方面则可能处于次要地位。Abstract: To investigate the roles of two Cpx component systems in the growth and virulence of Aeromonas hydrophila, we constructed the cpxRA gene cluster mutant Δcpx with the deletion of 57—1879 bp using suicide plasmid pRE112 as the vector. This procedure was based on fusion PCR and gene homologous recombination principles. Through electrophoresis and fluorescence quantitative PCR, the partial deletion of the cpxRA gene cluster in the mutant was confirmed. Then we compared the differences between mutant and wild strains in biological characteristics including growth, biofilm formation, stress tolerance and virulence. The results showed that the mutant had no significant difference with the wild strain in morphology, growth, biofilm formation and virulence. The main differences existed in the response to high osmotic pressure, SDS (Sodium dodecyl sulfate), EDTA (Ethylene Diamine Tetraacetic Acid) and polymyxin B stimulation. This study reveals that the Cpx system of Aeromonas hydrophila is involved in the response to external stimulus factors, and plays a relatively minor role in virulence.
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Key words:
- Aeromonas hydrophila /
- cpxRA /
- pRE112 /
- Stress tolerance
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图 9 斑马鱼浸泡攻毒累积存活曲线
Figure 9. The survival curve of zebrafish attacked by Aeromonas hydrophila strains soak
表 1 本研究中使用的引物
Table 1. Primers used in this study
引物Primers 序列Sequences (5′—3′) 用途Usage cpx-5O CGC TCTAGACAGGTCGGAGCGGTAGT Amplification of up-stream of gene cpxR:F1 cpx-5I CGAAAGAAGGGCAGGAACTCGGTGAGCAACTGGGT cpx-3O CCC GGTACCTTGGTGGTACAGGCGAAT Amplification of down-stream of gene cpxA:F2 cpx-3I ACCCAGTTGCTCACCGAGTTCCTGCCCTTCTTTCG cpx-incheck-F TTGAGCAGGGAGGAGATG Identification of cpxRA mutant cpx-incheck-R TGTCATCCCACTCAAACCC cpx-outcheck-F AGACCTCCTCCTGACCT Identification of cpxRA mutant cpx-outcheck-R TGCACCGATTCATAGC PRE-check-F TTCGTCTCAGCCAATC Sequencing the recombinant plasmid pRE-Δcpx pRE-check-R TGGTGCGTACCGGGTTG com-F CCC AAGCTTGGTAATCAGCAGGGTGGC Construction of cpxRA complementary strain com-R GTGA GCATGCGAGTCTGCTCAGCCGATG 注: 引物序列下划线部分表示酶切位点 Note: The underline primer sequence represents restriction site 
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表 2 荧光定量PCR引物
Table 2. Primers used in qRT-PCR
引物编号
Primers引物序列
Sequences (5′—3′)编码基因
CDScpxR-F GCTGCTGGACGTGATGATGC CpxR cpxR-R CGTTGACCCGATCCTGGC cpxA_F GCTGTTGCTGGTGGTGGC CpxA cpxA-R AATCCTGATTGGGGTCTG 16s-F CAACCCCTGTCCTTTGTT 16S rRNA 16s-R TTTGGGATTCGCTCACTA alt-F TGCTGGAGCTGAGCTTTG Alt alt-R CTGTCCTTGAGGGAGTCG hly-F GATGGCATCGGTGGCATA Hly hly-R CGCTGGACGAAGAGTCGG aer-F TAACCCGGCCCCATTATT Aer aer-R CGGCAGAGCCCGTCTATC env-F TCTCCTATGCCACCTTCT EnvZ env-R TCGGCGTCTTCACTCAAC omp-F TCAGGCTGAAGTTCTCAC OmpR omp-R AATACAAGGTTCTGGTCG QseB-F GTCACGGGCGGTGAGGAT QseB QseB-R TCAAGAGCGAGGAGTTTG QseC-F GAGATGAGCCACCACAGC QseC QseC-R CAACAACGTCACCAAGGA dsbA-F TTCCTTGCTGCCATGCTG DsbA dsbA-R GTTGGGCGCTACCGGTCT RpoE-F TTCGCCACCGCCATAGTA RpoE RpoE-R CGCAGGAGGCATTCATCA ExbD1-F CTGCTTGTCTGGCTGGTT ExbD1 ExbD1-R TGGTGCTGCTCATCGTCT b561-F GAGCATCAGGGGGGAGAG B561 b561-R ATGGAGTTCAGGGACATC FimA F AAGTGGGGAAGAGATCGTG FimA FimA R ATAAAGTCGGCGGAGGCAT PilA F GAATTGATGATCGTGGTCG PilA PilA R CGCTGTTGCTATGTTTGCC ycfS F GTCGATCCCTATTTGCCAA YcfS ycfS R CACCATCACCTCGTTTTTA HK-F CGCAGCAGAGCCATCCACAA Histidine HK-R AGTCCGCCGATCCACCACAT kinases
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表 3 斑马鱼腹腔注射累积死亡情况
Table 3. The cumulative death of zebrafish injected A. hydrophila strains by intraperitoneal
菌株
Strain菌液浓度(cfu/尾)
Concentration累积死亡数
Cumulative death number累积死亡率
Cumulative mortality rate (%)1 2 3 4 5 6 7 Wild 6450 1 14 15 15 15 15 15 100 3225 0 12 13 13 13 13 13 86.67 1613 1 6 12 12 12 12 12 80 807 0 7 7 7 7 8 8 53.33 Δcpx 6600 1 13 15 15 15 15 15 100 3300 3 12 13 14 14 14 14 93.33 1650 2 7 11 11 11 11 11 73.33 825 0 9 9 9 9 9 9 60.00
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