文章摘要
不同培养条件下金黄色葡萄球菌表型可塑性研究
Phenotypic Plasticity of Staphylococcus aureus under Different Culture Conditions
  
DOI:doi:10.3969/j.issn.1005-7021.2019.04.002
中文关键词: 表型可塑性  金黄色葡萄球菌  SNP  双变量全基因组关联分析  相对表达量
英文关键词: phenotypic plasticity  Staphylococcus aureus  SNP  bi-variable genome-wide association study (GWAS)  relative expression
基金项目:中央高校基本科研业务费专项(2017JC05);国家自然科学基金青年科学基金项目(51108029)
作者单位
朱璟 北京林业大学 生物科学与技术学院北京 100083 
王峥鉴 北京林业大学 生物科学与技术学院北京 100083 
张佐然 北京林业大学 生物科学与技术学院北京 100083 
李金婷 北京林业大学 生物科学与技术学院北京 100083 
梁雅静 北京林业大学 生物科学与技术学院北京 100083 
金一 北京林业大学 生物科学与技术学院北京 100083 
何晓青 北京林业大学 生物科学与技术学院北京 100083 
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中文摘要:
      表型可塑性在生物界普遍存在,但在微生物领域的研究相对较少,利用分子标记技术研究微生物表型可塑性方法也鲜有报道。用1株大肠埃希菌与45株金黄色葡萄球菌混合培养营造竞争环境,测定其生长量并与相同起始浓度单独培养的金黄色葡萄球菌生长量做GWAS对比,得到显著SNP位点。分析SNP位点中与金黄色葡萄球菌生长变化相关的基因表达量变化情况,研究其与金黄色葡萄球菌表型可塑性的关联性。以45株金黄色葡萄球菌在两种模式下生长量及全基因组测序结果为基础,利用双变量全基因组关联分析(Genome-wide association study,GWAS)法筛选与金黄色葡萄球菌生长量显著相关的单核苷酸多态性(Single nucleotide polymorphism,SNP)位点,定位这些SNP位点对应的金黄色葡萄球菌基因,从这些基因的功能中筛选与金黄色葡萄球菌生长变化相关的部分并汇总分析。之后选取其中关联性较强的scdA基因序列为模板设计引物,用实时荧光定量PCR(Real-time quantitative PCR,qPCR)的相对定量法,选用16S rDNA为内参基因,测定两种培养环境下基因的mRNA相对表达量,统计分析差异性。结果显示,与大肠埃希菌混合培养的金黄色葡萄球菌受其影响生长量显著降低。GWAS在12个取样点共检测出415个显著SNP位点,筛选后对scdA基因两种培养条件下的相对表达量进行测量并统计分析,结果显示无论251050位点碱基为A或G,scdA基因在混合培养中的相对表达量都显著高于单独培养中的表达量。测序结果表明整个培养过程中scdA基因型保持不变,而两种培养环境下金黄色葡萄球菌scdA基因相对表达量的变化反映了表型的差异。为了应对环境压力,金黄色葡萄球菌SNP251050对应的scdA基因在混合培养环境下显著提高了表达量,以维持自身在培养体系中的稳定性。这表明金黄色葡萄球菌能够依据环境变化在广义生理表型方面主动做出对环境信号的多样性响应,也可认为是金黄色葡萄球菌表型可塑性在环境变化下的反映。
英文摘要:
      Phenotypic plasticity is ubiquitous in the biosphere, but relatively few studies on the microbial field, and there are few reports about using molecular marker technology to study microbial phenotype plasticity. One E. coli strain and 45 Staphylococcus aureus strains were adopted and mixed together and cultured to construct a competitive environment, and determined their growth volume and did GWAS (genome wide association study) comparison with the growth volume of the same initial concentration of the S. aureus cultured alone, and obtained significant SNP sites. Analyzed the expression variation correlated with the growth variation of S. aureus within the SNP sites, and studied the precision of their phenotypic plasticity of S. aureus. Based on the growth volume of 45 S. aureus strains under two modes and the sequencing results of full genotype, and adopted bi variable GWAS to screen the SNP sites that significantly correlated to growth volume of S. aureus,and localized the S. aureus genes corresponding to these SNP sites, and screened from the functions of these genes that correlated to the part of the growth variation of S. aureus to summarize and analyze. Then selected scdA sequence with relatively strong precision among them as template to design primer, using relative quantitative method of real time fluorescence quantitative PCR (qPCR), selected and used 16S rRNA as internal reference gene to measure the gene mRNA expression under two culture environment, then statistically analyzed the differences. The results showed that the growth volume of S. aureus mixed cultured with E. coli significantly declined. 415 obvious SNP sites were checked out by GWAS at 12 sampling sites, and after screening the expression of scdA gene under two culture conditions was measured and analyzed statistically. The results showed that regardless of base of A or G of 251050 site, the relative expression of scdA gene in mixed culture was significantly higher than that in the single culture. Therefore, the sequencing results showed that during the whole culturing process the genotype of scdA remained unchanged. In order to respond the environmental stress, the scdA gene corresponding to S. aureus SNP25150 expression under the mixed culture environment was significantly boosted, to maintain its own stability in the culture system. This suggested that S. aureus could make responses by taking the initiative in diversity according to the environmental signals at the respects of physiological phenotype in broad sense, this could also considered as the reflection of phenotypic plasticity of S. aureus under environmental changes.
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