siRNA分子干扰对副干酪乳杆菌群体效应相关基因表达的影响

【作者】 王洋；

【导师】 平文祥；

【作者基本信息】 黑龙江大学 ， 微生物学， 2014， 硕士

【摘要】 副干酪乳杆菌(Lactobacillus paracasei)HDI.7是从乳酸酸菜发酵液中分离出的一株乳酸菌。该菌发酵过程中产生的细菌素Paracin1.7,可有效抑制多种革兰氏阳性菌(G+)、革兰氏阴性菌(G-)及酿酒酵母的生长。本研究探讨了L.paracasei HD1.7群体效应相关基因(prcK、prcR)与其产细菌素之间的关系,为研究该菌群体效应相关基因功能以及群体效应作用机制奠定基础,从机理上揭示细菌素的产生与群体效应的关系。本研究首先利用RNA干扰技术,根据L.paracasei HD1.7基因组中存在的群体效应相关基因prcK和prcR片段,设计合成8个特异性小干扰RNA分子(small interference RNA,siRNA)及1个5’-FAM标记的非特异性siRNA。通过电转化方法将合成的siRNA分子转入试验菌株L.paracasei HD1.7中,结果表明,转入5’-FAM siRNA分子的菌体细胞中可见绿色荧光物质,说明电转化方法可有效的将外源siRNA分子导入细胞中。通过荧光定量PCR方法对培养24 h的基因沉默突变株进行prcK和prcR相关基因mRNA表达水平的测定,结果发现,分别针对prcK、prcR基因设计的8个特异性siRNA分子中仅siRNA-K4、siRNA-K5和siRNA-R4、siRNA-R6有明显抑制基因表达的作用。抑菌试验测定显示,两个prcK基因沉默突变株及两个prcR基因沉默突变株的抑菌能力均比原始菌株弱,分别比原始菌株降低22.48%、20.61%、21.43%和20.70%,说明prcK和prcR基因的沉默对突变株细菌素的产生具有一定的影响。为进一步证明prcK和prcR基因在调节细菌素生成中的作用,本研究利用过表达技术,以GenBank上已发表的prcK和prcR序列为参考序列,应用Primer 5软件设计特异性引物,将His标签基因序列融合于prcK基因和prcR基因的5’端,通过PCR方法,扩增His-prcK基因和His-prcR基因,并分别将其克隆到pMD18-T载体上,经酶切验证以及序列测定结果分析,证明获得了含有His-prcK融合基因和His-prcR融合基因的阳性重组质粒pMD18-T-prcK和pMD18-T-prcR。将His-prcK基因和His-prcR基因亚克隆至表达载体pRSFDuet-1中,经酶切鉴定,证明成功获得了重组过表达载体pRK-tet、pRR-tet。将这些质粒以电转化法导入试验菌株L.paracasei HD1.7中。经过四环素抗性筛选获得prcK和prcR基因过表达突变株。通过荧光定量PCR方法对培养24 h的基因过表达突变株进行prcK和prcR基因mRNA表达水平的测定,结果表明,prcK和prcR基因过表达突变株比原始菌株相应基因表达水平提高23.28%和31.40%。利用SDS-PAGE和Western-blotting成功检测到prcK、prcR基因蛋白的表达,且表达量比原始菌株提高119.37%和131.04%。抑菌试验显示,prcK和prcR基因过表达突变株均比原始菌株的抑菌能力提高17.86%和20.69%,说明prcK和prcR基因的过表达对突变株细菌素的生成产生了一定的影响。本研究通过构建prcK、prcR基因沉默及过表达突变株,并对突变株的抑菌效果进行测定,发现prcK、prcR基因的沉默可使突变株产细菌素的能力下降,而prcK、prcR基因的过表达可使突变株产细菌素的能力提高,说明prcK、prcR基因是L.paracasei HD1.7产细菌素的相关基因。经前期研究发现prcK、prcR基因与所编码的蛋白质分别与群体效应系统中HPK家族中HPK10亚家族和反应调节因子LytR/AlgR家族十分相似。综合以上得出结论,prcK和prcR基因是调控L.paracasei HD1.7细菌素生成的群体效应三组分调节系统中的组氨酸蛋白激酶编码基因和反应调节因子。本研究为今后L.paracasei HD1.7细菌素可控系统的构建,以及产细菌素条件优化等方面的研究提供理论依据,使L.paracasei细菌素及其合成途径具有更广阔的开发前景和应用空间,更多造福于人类。更多还原

【Abstract】 Lactobacillus paracasei HD 1.7 was isolated from Chinese sauerkraut fermentation broth.L.paracasei HD 1.7 secreted a kind of bacteriocin Paracinl.7 during fermentation process.The Paracinl.7 could inhibit the growth of some G+bacteria,some G-bacteria and Saccharomyces cerevisiae.We investigated the relationship between the L.paracasei HD1.7 quorum sensing related genes,i.e.prcK and prcR and the Paracinl.7 production.This research could be a potential basis for the exploration of the function of prcK and prcR and the mechanism of L.paracasei HD 1.7 quorum sensing.This research aimed to reveal the relationship between the Paracinl.7 production and the quorum sensing.In this research,gene silence technology was used to explore the function of prcK and prcR of L.paracasei HD 1.7.According to the sequences of prcK and prcR,we designed eight specific siRNAs and one 5’-FAM siRNA.Electrotransformation technique was adopted to transform these siRNAs into L.paracasei HD1.7.The expression level of related genes in prcK and prcR silence mutant strains in the 24-hour culture were detected by real time PCR.The results showed that siRNA-K4,siRNA-K5,siRNA-R4 and siRNA-R6 significantly inhibited genes repression.Antibacterial tests were earried out to observe the antibacterial condition of mutant strrains.The antibacterial activities of mutant strains were lower than the original strain.The titer decrease by 22.48%,20.61%,21.43%and 20.70%,respectively.This result testified that the silence of prcK and prcR had an influence on paracin 1.7 production.Furthermore,gene over expression technology was used to explore the function of prcK and prcR.Specific primers were designed by Primier 5 according to the sequences of prcK and prcR derived from GenBank.Then his-tag was added to the 5’ end of the target gene.His-prcK gene and His-prcR gene were amplified and ligated to pMD18-T vector.Via the restriction enzyme digestion analysis and nucleotide sequencing,the positive recombinant plasmids pMD18-T-prcK and pMD18-T-prcR were obtained which contained His-prcK and His-prcR fusion gene,respectively.Then His-prcK and His-prcR were cloned to the expression vector pRSFDuet-1 and the recombinant expression vectors pRK-tet and pRR-tet were obtained.These constructed plasmids were introduced into L.paracasei HD 1.7 by electrotransformation.The mutant strains with over expression of prcK and prcR were selected by teracyline resistance screening.The level of over expression of the two genes in mutant strains in the 24-h culture was detected by real time PCR.The results showed that expression level of related genes in mutant strains were higher than that in the original strain by 23.28%and 31.40%respectively.His-prcK protein and His-prcR protein were successfully detected by SDS-PAGE and Western blotting.Expression levels of related proteins in mutant strains were higher than that in the original strain by 119.37%and 131.04%respectively.Antibacterial tests were done to observe antibacterial condition of mutant strains.The results demonstrated that the antibacterial activities of mutant strains were stronger than the original strain.The titer reached an increase of 17.86%and 20.69%,respectively.This data testified that the over expression of prcK and prcR had an influence on Paracin 1.7 production.By constructing and over expressing the silence mutant strains of prcK and prcR,as well as conducting the antibacterial tests,this study revealed that the antibacterial activities of silence mutant strains decreased and over expression mutant strains increased compared with that in the original strain.It suggested that prcK and prcR had n influence on Paracin 1.7 production.Preliminary reaserch found that prcK and prcR were similar with HPK10 subfamily of HPK and LytR/AlgR family respectively.Taking into account all the results,the work could testify that prcK and prcR was sensor kinase and response regulator.This research could provide foundation for the construction of regulation system and the optimization of bacteriocin production.It provided more broad development prospect,application space and could benefit human.更多还原