反向遗传学策略解析S. maltophilia DHHJ 菌株SMBP蛋白及其膜受体功能

As encrusted protein, feather keratin has the characteristics of no-dissolve, anti-decomposition and anti-pain, and it also cannot be dissolved in normal conditions. So feather keratin is very difficult to be deal with. And at the same time a lot of keratin materials, as wastes, are released in China, such as feathers from poultry farms, leather heels, and so on. To protect our environment and make full use of the resources，most of researchers will turn the wastes to treasures with biotechnology method because of its advantages of no pollution and easy to control. In our initial research，a novel kerationlytic bacterium, Stenotrophomonas maltophilia DHHJ had been isolated and identified (Cao et al, 2009). The bacterium strain is very well to degrade feather. Traditionally, it thinks enzymes are the key factor to degrade macromolecular material. Therefore, more than 50 keratin hydrolysis enzymes (keratinase) had been identified by different laboratories all over the world. But feather keratin can`t bedegraded by these enzymes in vitro. Another side, E.coli that transfer keratinase gene hasn`t the capability to degrade feather through different research in several laboratory. So, researchers think some other molecular works to degrade feather. We find a peptide binding protein, named SMBP, which is a key factor to degrade feather in S. maltophilia DHHJ. We would like to do some research based this: Firstly, the function of SMBP would be sure through RNA interference method. Secondly, SMBP would be sited through fusion expressed with GFP. In other side, keratin monomer was labeled with FITC. It would be observed about the molecular movement in the cultural liquid with bacteria. Thirdly, the membrane receptor of SMBP would be researched by molecular blotting method. The research would help people to understand the mechanism of feather keratin biodegraded. Further, it would provide a base to researcher improve the ability to degrade feather in bacterium.

禽类羽毛主要成份为角蛋白（86%-97%），是地球上最大的氮源废弃物之一，炼制产物用途广泛，生物炼制由于多种优点而成为目前潮流。但羽毛生物炼制的机理一直是该领域的难解点，因此炼制效率难以提高，限制了该工艺应用。申请人前期研究得到了羽毛角蛋白降解酶特征及基因序列，并发现了S. maltophilia DHHJ 菌株中与羽毛降解相关蛋白—肽链结合蛋白（SMBP），本项目研究在此基础上，利用RNAi技术分析SMBP蛋白对角蛋白降解作用，包括了对酶表达及角蛋白降解效率的影响；同时利用荧光定量PCR技术初步确定角蛋白进入细胞的通道蛋白。从而进一步阐明角蛋白降解的分子过程，为后续菌种改造，角蛋白生物炼制生化机制的全面解析奠定基础，同时为角蛋白作为新型氮源利用具有重要的实用价值。

羽毛角蛋白降解机制研究为提高炼制效率，羽毛变废为宝提供理论基础。本课题组分离鉴定出一高效降解羽毛细菌（S.maltophilia DHHJ），并发现其降解羽毛由胞外及胞内因子共同作用，胞外本底表达的酶因子降解羽毛为角蛋白单体，其激活胞内相关基因表达系统。在前期工作基础上及自然基金资助下完成以下工作：.分别以化学水解法制备及外源表达的角蛋白单体（~10 kD）及其酶解后的片段为诱导源，以不添加角蛋白为阴性对照，添加羽毛粉为阳性对照。将由异硫氰酸荧光素（FITC）标记的角蛋白与S.maltophilia DHHJ菌株共培养，分别向其中加入不同浓度的BSA、DNA、ATP、不同盐离子等共培养，测定了72 h内S. maltophilia DHHJ荧光强度及角蛋白酶活。结果发现DHHJ菌角蛋白酶活性与荧光结合相一致，即角蛋白单体结合越多，酶活越高，表明角蛋白单体激发了羽毛降解酶基因表达过程。.利用RNA-seq分析S.maltophilia DHHJ在分泌角蛋白酶时诱导及非诱导个体转录组。从中挑选出6个候选基因并与蛋白组分析结果比较，通过qRT-PCR分析基因在表达量，跨膜转运蛋白、ABC转运蛋白以及氨基酸跨膜相关的基因富集较为显著。对其进行KEGG代谢通路分析，其中氨基酸代谢、细菌分泌系统以及ABC转运体系等通道都有非常活跃的上调表达。细菌与角蛋白共培养，角蛋白作为营养源与细菌结合并通过蛋白转运系统被摄入细胞内，从而刺激细菌开始分泌角蛋白酶，产角蛋白酶活性。.对SMBP外源表达且其与角蛋白单位结合时，发现与膜上ABC系统相互作用，分别激发了ABC底物结合蛋白、多肽ABC转运透过酶等蛋白，该结果与前面ATP促进蛋白结合及酶基因表达相一致，初步说明ABC体系参与羽毛降解的基因起始识别与信号转换过程。.本项目执行过程中，研究了其代谢规律及体内外对酶降解羽毛的促进作用，该结果不仅为微生物降解羽毛分子机理全面解析奠定了基础，而且为微生物精准利用氮源研究提供一条新的线索。