铅结合蛋白PbrR的大肠埃希菌外膜展示株构建及体内定植初步研究

目的 构建铅特异性结合蛋白PbrR的大肠埃希菌外膜展示株，并考察其在小鼠体内的定植及外膜展示的重组蛋白在消化道内环境耐受能力。方法 全基因合成嵌合蛋白Lpp-OmpA编码序列，将其插入表达载体pET-21a，构建外膜蛋白展示载体pLOA。全基因合成PbrR编码序列，将其插入pLOA，构建PbrR外膜展示质粒pLOA-pbrr。转化表达宿主大肠埃希菌BL21（DE3）pLysS，IPTG诱导表达，15% SDS-PAGE及Western blot分析表达情况。考察PbrR外膜展示株在人工肠液中的铅吸附能力及在人工胃液中的存活能力。KM小鼠经口连续7 d给予诱导后的展示株，终止染菌后，继续饲喂至30 d，稀释涂平板法检测第7、15、30天小鼠粪便中重组菌含量，免疫印迹法检测第7和第15天小鼠粪便中的重组蛋白残留。结果 基于Lpp-OmpA展示载体，成功构建了PbrR的外膜展示质粒。融合蛋白Lpp-OmpA-PbrR-His tag得到了高水平表达，展示株在人工肠液中表现出了显著升高的铅离子吸附能力。展示株表现出了一定的胃酸耐受性，经口染菌后可定植于小鼠肠道，外膜展示的重组融合蛋白对消化道内环境表现出较好的抵抗能力。结论 大肠埃希菌PbrR外膜展示株在体外模拟肠道环境中表现出了较强的铅富集能力，体内可定植小鼠消化道，外膜展示蛋白也有较好的消化液耐受能力。本文为进一步基于动物模型的生物吸附选择性驱铅的研究提供基础资料。

PbrR大肠埃希菌外膜展示株体内外生长特性

目的考察Pbr R大肠埃希菌外膜展示株在不同条件下的体内外生长特性,为其体内定植驱铅能力的研究奠定基础。方法 15%SDS-PAGE分析不同浓度诱导剂L-阿拉伯糖诱导展示菌p BAD-loa-pbrr/DH10B的重组蛋白表达情况;铅平板敏感实验对比重组菌诱导及非诱导条件下在含铅平板上的菌落形成能力;体外培养实验考察诱导剂的加入对重组菌生长能力及耐铅能力的影响;通过混合共培养实验对比诱导条件下重组菌与空宿主菌的竞争生长能力;考察重组菌6 h内的胃酸耐受性;考察诱导剂及染菌方式对重组菌小鼠体内定植的影响。结果低至0.002%的L-阿拉伯糖即可诱导重组蛋白的高效表达;Pbr R展示菌对铅表现出了一定的耐受力;诱导剂引入后重组菌生长能力降低;重组菌对胃酸有较好的抗性,经口染菌方式重组菌可定植于小鼠肠道,但摄入L-阿拉伯糖水后,粪便中重组菌快速丢失,通过连续染菌方式可维持其定植水平。结论重组菌体内外培养实验结果表明,基于商品化的诱导型表达载体构建的展示菌难以表达重组蛋白的同时实现小鼠体内的稳定定植。

PbGIF1 promoting cell-proliferation in pear fruit is transcriptionally activated by Pb RR1

As a cell proliferation regulator involved in wide biological processes in plants,GRF-INTERACTING FACTOR(GIF)controls different tissues development.However,whether GIF participates in fruit development remains unclear.According to transcriptome data,we identified PbGIF1was highly expressed during fruit development in cytokinins induced parthenocarpy pear.In the present study,the biofunction of PbGIF1 was initially verified.Overexpression of PbGIF1 promoted fruit size of transgenic tomato.The size of flesh fruit was not affected by cell expansion but the cell proliferation was promoted by overexpressing Pb GIF1.The accelerated cell proliferation process was also observed in PbGIF1-overexpressed transgenic pear fruit calli.The transcriptional regulation of cytokinins on PbGIF1 was further confirmed by exogenous CPPU treatments in pear fruitlets.To investigate the underlying mechanism,the cytokinins-responded factor,PbRR1,was further focused on.The results of Yeast-one-hybrid assay suggested that PbRR1 can bind to the promoter sequence of PbGIF1.The transcriptional activation of PbRR1 on PbGIF1 was also confirmed by Dual-Luciferase assays.Taken together,the results showed that cytokinins control pear fruit development via the transcriptional activation of PbGIF1 by PbRR1.