辽宁慢生根瘤菌抗铜基因的克隆及功能分析

发布时间：2018-01-23 06:35

  本文关键词： 辽宁慢生根瘤菌 Tn5-pRL1063a 铜抗性 操纵子　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

【摘要】：土壤的铜污染会给其中微生物造成一定的选择压力,根瘤菌要保证自身的存活并且与宿主植物形成有效的共生固氮体系,必须采取一定的保护机制来抵抗高铜浓度造成的胁迫。分离自陕西宁强代家坝铜锌矿中的辽宁慢生根瘤菌Bradyrhizobium liaoningense CCNWSX0360对铜的最大耐受浓度为2.0 mM。本文利用转座子Tn5-pRL1063a对B.liaoningense CCNWSX0360进行随机突变,建立了库容为17247的突变体库。铜敏感性筛选获得6株突变株Bln-d、Bln-163、Bln-c、Bln-29、Bln-32和Bln-54。采用质粒挽救法确定Tn5的插入位点,结果显示Bln-d和Bln-163中Tn5插入到同一个基因的不同位置,相似性比对鉴定为编码重金属转运P1B-type ATPase的基因,命名为cueA,其余4个突变体中Tn5分别插入到基因copA(编码多铜氧化酶)、ctpA(编码羧基末端处理酶)、tolC(编码TolC家族蛋白)和lptE(编码脂多糖转运分子伴侣)内部。重金属耐受性分析结果显示:Bln-d、Bln-163、Bln-c专一对铜敏感,说明cueA和cop A特异地介导菌株对铜的抗性,在保护细菌免受铜的毒害中起关键作用;除Cu以外,Bln-32、Bln-54和Bln-29不同程度地对其它重金属敏感,说明ctpA、tolC和lptE通过间接途径赋予根瘤菌铜抗性。根据上述基因的功能注释得出:在B.liaoningense CCNWSX0360中至少存在3种抗铜机制:跨膜转运机制(cueA和tolC)、氧化/还原机制(copA)和膜屏障保护机制(ctpA和lptE),共同赋予细菌抵抗铜胁迫的能力。根据跨膜金属结合基序及标签序列信息本研究将cueA编码的蛋白CueA归为Cu+/Ag+转运P1B-type ATPase。CueA的N端的金属结合基序为两段His-rich区,而不是典型的GXXCXXC基序。利用同源重组构建了突变体ΔcsoR和ΔcueA,铜耐受性测试结果显示:ΔcueA和cueA::Tn5的铜敏感程度相同,对铜的最大耐受浓度(MTC)为0.8mM,显著低于野生型;互补cueA基因能够回复突变体到野生型菌株的抗性水平。qRT-PCR和lacZ融合表达分析结果表明:Cu2+显著诱导cueA的表达且表达水平依赖于Cu2+的浓度;一价铜离子螯合剂BCS能够消除(减小)Cu2+对cueA的诱导和对细菌的毒害。结合铜耐受性测试、qRT-PCR以及lacZ融合表达分析结果证实了cueA通过向细胞周质空间转运一价Cu+来维持根瘤菌的铜抗性。运用qRT-PCR和lacZ融合表达分析技术检测了野生型、突变体ΔcsoR以及互补菌株ΔcsoR(csoR)中csoR和cueA的表达水平,结果表明:缺失csoR引起基因cueA和csoR高水平组成型表达;互补csoR恢复依赖Cu2+的诱导表达,证明了CsoR利用去阻遏机制负调节csoR和cueA。实验结果还表明:突变csoR显著增加了菌株对Cu、Zn和Cd的抗性,并且这3种金属离子均能诱导基因csoR和cueA的表达;在Escherichia coli GG48中表达CueA能够提高该菌对Zn和Cd抗性,说明cueA不仅能够介导Cu+而且介导Zn2+和Cd2+的抗性。此外,N端截短实验发现:缺失N端的His-rich区丧失CueA的部分铜抗性功能,完全丧失在CueA的Zn/Cd抗性功能,表明N端的His-rich区在CueA的抗铜功能中发挥重要作用但不是必须的,而在抗Zn和Cd的功能中是绝对需要的,具体作用还不清楚,可能通过金属离子结合位点来缓冲细胞内的金属离子浓度。通过共接菌实验比较野生菌和突变体?cueA的竞争结瘤能力,得出根瘤菌的铜抗性能够赋予其在铜胁迫下的结瘤能力。基因突变以及铜耐受性分析结果显示:cop操纵子中6个基因均专一对铜敏感,其中copA和copB是整个系统的核心组分,二者需要同时存在才能发挥作用;新基因2212和2213能够赋予cop操纵子的完全的铜抗性;copC和cus F则对菌株的耐受性贡献不大,推测这6个基因编码的蛋白可能相互作用形成介导铜抗性的符合体。这6个基因在不同铜浓度胁迫下具有相同的表达趋势,即在较低Cu2+浓度下,随着Cu2+浓度的升高各个基因的转录水平逐渐增加,在0.625 mM Cu2+浓度下表达量均达到最大,在高浓度下则转录水平下降。综合共转录验证实验及5’RACE实验得出这6个基因为一个转录单元,受同一个启动子启动转录,转录起始点为基因2213起始密码子上游第26个碱基“G”。启动子活性分析以及lacZ融合表达分析结果表明2213与2212之间的间隔序列在调节操纵子的表达起着关键作用,但具体调控机制还未研究不清楚。生物信息学分析数据显示:cop操纵子可能为一个进化上较为古老的铜抗性操纵子。与单独敲除cueA或copA相比,同时敲除这两个基因菌株更为敏感,说明cueA和cop操纵子两个独立的系统,共同保护细胞免受铜离子的毒害。
[Abstract]:The soil copper contamination caused by microorganisms which will give certain selection pressure, rhizobia to ensure their own survival and the formation of host plant symbiotic nitrogen fixation system effectively, must take protective mechanism to stress resistance caused by high copper concentration. Isolated from Shaanxi Ningqiang daijiaba copper zinc ore in Liaoning Bradyrhizobium Bradyrhizobium liaoningense CCNWSX0360 maximum tolerated concentration of copper is 2 mM. by using Tn5-pRL1063a transposon random mutation of B.liaoningense CCNWSX0360, a capacity of 17247 of the mutant library. Copper sensitivity screening 6 strains Bln-d, Bln-163, Bln-c, Bln-29, Bln-32 and Bln-54. using plasmid rescue method to determine the Tn5 insertion site, the results showed Bln-d and Bln-163 Tn5 into different positions of the same gene, similarity comparison identified as encoding P1B-type heavy metal transporter ATPase The gene, named cueA, the remaining 4 mutants in Tn5 were inserted into the copA gene (encoding multicopper oxidase), ctpA (encoding the carboxyl terminal processing enzyme (tolC), encoding TolC protein family) and lptE (encoding transporter lipopolysaccharide molecular chaperone). Heavy metal tolerance analysis showed: Bln-d, Bln-163, Bln-c specific the copper sensitive, cueA and cop A specifically mediate resistance to copper, plays a key role in the protection of the bacteria from copper toxicity; except Cu, Bln-32, Bln-54 and Bln-29 in different degrees of other heavy metal sensitive, that ctpA, tolC and lptE by indirect way gives rhizobia copper resistance. According to the functional annotation of these genes showed that: there are at least 3 kinds of copper resistance mechanism in B.liaoningense CCNWSX0360: transmembrane transport mechanism (cueA and tolC), oxidation / reduction mechanism (copA) and membrane barrier protection mechanisms (ctpA and lptE), to give bacterial resistance to copper Stress. According to the transmembrane metal binding motif and tag sequence information on the cueA encoding protein CueA belongs to the Cu+/Ag+ ATPase.CueA transfer P1B-type N end of the metal binding motif for the two His-rich area, rather than the typical GXXCXXC motif. We constructed a mutant csoR and cueA homologous recombination. The test results showed that copper tolerance of Delta cueA and cueA: copper Tn5 sensitivity, the maximum tolerated concentration of copper (MTC) 0.8mM, was significantly lower than that of the wild type; complementary cueA gene can recover the mutant to wild type strain resistance level of.QRT-PCR and lacZ fusion expression analysis results showed that the concentration of cueA and the expression depends on the level of Cu2+ was significantly induced by Cu2+; copper ion chelating agent BCS can eliminate (decrease) induced by Cu2+ on cueA and on bacterial toxicity. Combined with copper tolerance test, qRT-PCR and lacZ fusion expression analysis results Confirmed through cueA to the periplasmic space transport of monovalent Cu+ to maintain the rhizobia of copper resistance. Using qRT-PCR and lacZ fusion analysis technique was used to detect the expression of the wild type and mutant and complementary strain Delta csoR Delta csoR (csoR) expression in csoR and cueA. The results showed that the expression of cueA and csoR caused by the high level group the lack of forming csoR; complementary induced expression of csoR dependent Cu2+ recovery, proved by CsoR derepression mechanism of negative regulation of csoR and cueA., the experiment results also indicated that the mutation of csoR significantly increased the resistance of Zn strains to Cu, and Cd, and the expression of the 3 kinds of metal ions were induced by csoR gene and cueA CueA can be improved; the bacteria of Zn and Cd expression in Escherichia coli GG48 resistance, cueA resistance and can not only mediate Cu+ mediated Zn2+ and Cd2+. In addition, found N truncated experiment: part of the copper loss of N end His-rich CueA anti loss zone Complete loss of function, function in Zn/Cd resistant CueA, showed that the N side of the His-rich area in CueA copper resistance function plays an important role but not necessary, and in the anti Zn and Cd function is absolutely necessary, the specific function is not clear, the concentration of metal ions may buffer cells via metal ion binding sites. By CO inoculation experiments comparing wild strains and mutants? Competitive nodulation ability of cueA, the rhizobia can give its Cu resistance under the stress of copper nodulation ability. Gene mutation and copper tolerance analysis results show that the cop operon in 6 genes specific for copper sensitive copA and copB is the core component of the whole system, the two need to exist at the same time can play a role; completely new copper resistance gene 2212 and 2213 to give the cop operon and cus F copC; the strain tolerance contributed little to push the 6 test The gene encoding protein may be formed with copper resistance mediated interactions. These 6 genes have the same expression trend in different concentrations of copper stress, even at a low concentration of Cu2+, with elevated levels of transcription of each gene Cu2+ concentration increased gradually, reached the maximum amount of expression at 0.625 mM concentration of Cu2+ then, the transcription level decreased at high concentration. The comprehensive cotranscription validation experiment and 5 RACE experiment shows that the 6 gene is a transcription unit, by the same promoter transcription, transcription start point for 2213 genes upstream of the start codon twenty-sixth nucleotides G. The promoter activity of lacZ and fusion expression analysis showed that the interval between 2213 and 2212 sequence plays a key role in regulating the expression of the operon, but the specific regulatory mechanism has not been studied clearly. Bioinformatics analysis data show that: the cop operon may be a A more ancient copper resistance operon is more sensitive than knocking out cueA or copA individually. It is more sensitive to knock out the two genes, indicating that two independent systems of cueA and cop operon protect the cells from copper toxicity.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q939.114
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