乳酸乳球菌耐酸长链非编码ncRNA263的调控机制研究

Our previous work elucidated the relationship between acid tolerance capacity and cell wall in L. lactis. Deep sequencing of L lactis under acid stress showed that the transcription of 19 ncRNA is up-regulated after acid stress in which over-transcription of 6 ncRNA can obviously improve the acid tolerance and nisin yield of the strain. Among them, one long chain ncRNA 263 was predicted to control the biosynthesis of cell wall. However, the mechanisms of how ncRNA 263 is regulated and how it regulate the downstream genes are still unclear. This study is designed to obtain the transcriptional factors which could interact with the promoter of ncRNA 263, and try to further investigate the up-regulation mechanism by expression and EMSA. Furthermore, the regulatory role of ncRNA 263 under acid stress could be elucidated by some methods such as investigating the target genes of ncRNA 263 in E.coli. This program provided the important guiding for understanding regulation mechanism of acid-resistant long chain ncRNA263.

前期工作表明，乳酸乳球菌的耐酸能力与细胞壁厚度有非常密切的关系。乳酸乳球菌酸应激后深度转录组测序和荧光定量PCR分析表明，乳酸乳球菌酸F44中有19个ncRNA上调，过表达结果表明6个ncRNA可以显著提高菌株的耐酸能力及nisin生产能力。ncRNA 靶标预测表明，其中一个长链ncRNA263可以控制细胞壁的合成，但ncRNA263在酸应激过程中如何被调控？其如何调控细胞壁的合成机制还不清楚。本课题拟调取酸应激过程中与长链ncRNA263转录起始区结合的蛋白，并进一步通过表达及EMSA验证，了解该ncRNA的转录起始机制。另外，通过在大肠杆菌中异源表达验证该ncRNA可能的细胞壁合成靶标基因，确定其调控机制。本课题对于深入了解耐酸长链ncRNA263调控机制具有非常重要的指导意义。

前期工作表明乳酸乳球菌的耐酸能力与细胞壁厚度有非常密切的关系，过表达ncRNA263可显著提高菌株耐酸能力，预测发现其可能调控细胞壁合成。本项目对ncRNA263的功能进行了深入研究，包括通过调控细胞壁细胞壁合成以及影响菌株的酸胁迫能力，并通过靶标的预测与验证，寻到到了其直接调控靶标murA、murC和uxaC；此外，通过蛋白质组学研究发现，ncRNA263还显著影响核苷酸代谢和RNA加工过程，较全面的总结了ncRNA263对乳酸乳球菌F44的调控机制。通过DNA pull down和EMSA等方法，初步找到了ncRNA263的潜在转录调控因子RmaE和0595。同时，对前期基于转录组深度测序发现的与酸耐受相关的sRNA，包括sRNA s015、c277、s042、s013和s087，进行了鉴定并确定了它们响应乳酸乳球菌F44的酸胁迫的功能，通过靶标预测与荧光报告系统体内验证，分别确定了它们各自的直接调控靶标，较为深入的解析了sRNA s015、c277、s042、s013和s087的耐酸调控机制。本研究确定了调控ncRNA转录的转录调控因子和ncRNA的调控靶点，并从非编码RNA角度较为全面地解析了乳酸乳球菌F44的酸耐受调控机制，对于深入了解非编码RNA调控机制具有重要意义，同时也为乳酸菌耐酸调控网络的构建奠定了基础。