烟酰胺提高芽孢杆菌杀线虫活性的机制研究

Plant-parasitic nematodes are important pathogen second only to fungi. As nematocidal bacteria, Bacillus are important resource for biological control of nematodes. Our research team reported a new "Trojan horse" mechanism of bacterial pathogenesis against nematodes in Bacillus nematocida B16. But it is not known how does B16 regulate this "Trojan horse" infect process. In our previous study, it was found that nicotinamide (NAM), an inhibitor of protein deacetylase, improved the nematotoxic activity against Caenorhabditis elegans remarkably. This suggests that protein acetylation participate in regulating "Trojan horse" process of killing nematode. In order to elucidate the mechanism of improvement of nematotoxic activity against C. elegans by NAM, we plan to identify the modifications of lysine acetylation by proteome-wide analysis, and find out proteins related to nematocidal process. Whereafter, some key proteins will be selected to study their functions in nematocidal process, and to study the regulation mechanism of function by proteins's acetylation. And furthermore, by using SILAC technique, analyse variation on acetylation level of these key proteins arosed from the addition of NAM, resolve the correlation between variation on acetylation and improvement of nematotoxic activity by NAM. Our research will elucidate the mechanism how NAM improve the nematotoxic activity of B16 by regulating the acetylation of some key proteins. Our results will provide a basis to illustrate the regulation mechanisms of "Trojan horse" process of killing nematode by B16, and provide theoretical guidance for developing efficient anti-nematode bacterium.

作物寄生线虫已成为仅次于真菌病害的第二大作物病害，以芽孢杆菌为代表的杀线虫细菌是重要的线虫生防资源。申请人所在研究团队报道了杀线虫芽孢杆菌B16 侵染线虫的"特洛伊木马"新机制，但B16如何对"特洛伊木马式"的杀虫过程进行调控，目前还不清楚。申请人前期研究发现，蛋白质去乙酰化酶抑制剂- - 烟酰胺能够显著提高B16对线虫的毒杀活性，提示蛋白质乙酰化可能参与B16杀线虫的调控。为揭示其调控机制，本研究拟通过乙酰化蛋白质组测序，得到乙酰化蛋白质组数据，分析数据得到杀线虫相关蛋白的乙酰化修饰谱，选取关键蛋白质研究其在杀线虫过程中的功能以及乙酰化对其功能的调控机制，并通过SILAC技术定量分析蛋白乙酰化水平的变化，解析乙酰化水平变化与杀线虫活性提高的相关性。通过上述研究阐明烟酰胺通过调控相关蛋白的乙酰化水平提高B16杀线虫活性的分子机制，为揭示B16杀线虫的调控机制和开发高效线虫生防菌剂奠定基础。

前期研究发现，烟酰胺（NAM）能够显著提高Bacillus nematocida B16 的杀线虫活性。本研究通过测定Bacillus nematocida B16的蛋白组和乙酰化化蛋白组数据，在蛋白组数据中发现：当添加烟酰胺（NAM）之后，33个蛋白的表达量上调，24的蛋白的表达量下调；B16的其中一个杀线虫毒力蛋白Bae16的表达量提高了数倍，western blot证实NAM确实提高了Bae16的表达量，这个结果很好地解释了NAM为何能够提高B16的杀线虫活性。在乙酰化蛋白组数据中，总共鉴定到了349个蛋白质的529个赖氨酸位点存在乙酰化修饰，对这些蛋白进行COG注释、KEGG代谢途径分析等生信分析之后发现，22个乙酰化蛋白参与了B16线虫引诱剂的合成，2个蛋白参与了毒力蛋白的分泌。对其中的411个赖氨酸位点的乙酰化水平进行了定量测定，发现在B16杀线虫的过程中，有18个位点的乙酰化水平上调，19个位点的乙酰化水平下调；添加NAM之后12的位点的乙酰化水平下调，44个位点的乙酰化水平上调。代谢途径富集分析发现，乙酰化水平受NAM影响的蛋白富集在5个氨基酸代谢途径上。本研究揭示了NAM通过提高毒力蛋白Bae16的表达量来提高B16杀线虫活性，也揭示了B16杀线虫相关的蛋白质确实存在乙酰化修饰，为后续研究乙酰化对B16和线虫相互关系的调控作用奠定了基础。