实验海洋生物学重点实验室知识库

杀鱼爱德华氏菌（Edwardsiella piscicida）是海水养殖鱼类的重要病原菌，由杀鱼爱德华氏菌引发的爱德华氏菌病给我国水产养殖业造成了巨大的经济损失。细菌非编码小RNA是近些年来发现的一种调控因子，在转录后水平调节基因表达，参与调控环境应答、群体感应（quorum sensing）、生物膜形成、运动性和致病性等生物学过程。目前对于杀鱼爱德华氏菌的感染机制，尤其是非编码小RNA（sRNA）调控的毒力机制仍有待研究。本研究通过对不同生长环境下（正常LB培养，酸性胁迫，缺铁胁迫和氧化胁迫）培养的杀鱼爱德华氏菌提取总RNA进行高通量测序，总共发现有148个sRNA表达，其中19个是已经注释过的sRNA，其它129个未曾报道。相比正常LB培养组，共有103个sRNA在3种胁迫环境下有显著差异表达。根据测序结果，分别选取在酸性条件和氧化条件下具有显著差异表达的sRNA（命名为sR082和sR012）进行深入研究。

首先，用qRT-PCR法分析了sR082和sR012在不同生长时期和不同环境胁迫压力下的表达量，结果表明，sR082在细菌生长早期和低pH条件下高表达；sR012在细菌生长对数期高表达。为了研究sR012和sR082生物学功能，利用同源重组法分别构建了sR082和sR012敲除株TXΔsR082和TXΔsR012，比较敲除株和野生株在低pH压力和氧化压力条件下的生长情况。发现TXΔsR082在pH 5条件下生长能力显著降低；TXΔsR012在氧化压力条件下生长能力显著降低。这些结果表明sR082与sR012分别与细菌适应酸性和氧化胁迫能力有关。

为分析sR082与sR012对细菌感染宿主的影响，本研究分别比较了TXΔsR082，TXΔsR012与野生株TXD1感染牙鲆细胞、组织和个体能力的差异。结果表明，与TXD1相比，TXΔsR082和TXΔsR012感染牙鲆FG细胞能力、胞内复制能力、组织侵染能力以及致死能力均显著下降。这些结果表明，sR082和sR012在杀鱼爱德华氏菌耐受宿主胞内酸性和氧化环境及感染等方面发挥重要作用。

为进一步了解sR012在杀鱼爱德华氏菌中的调控作用，利用TargetRNA2软件对sR012进行靶基因预测，并用qRT-PCR验证靶基因的转录水平。发现共有4个靶基因（ETAE-0114，ETAE-2575，ETAE-3156和ETAE-0911）在TXD1和TXΔsR012中有差异表达，这些结果表明sR012可能通过调控这4个靶基因适应宿主胞内氧化环境从而影响杀鱼爱德华氏菌的毒力。

Edwardsiella piscicida is a major pathogen of marine fish in aquaculture. Edwardsiellosis caused by E. piscicida leading to severe economic loss. In recent years, Small noncoding RNA (sRNA) of bacteria function as regulators of gene expression at the post-transcriptional level and play critical regulatory roles in major biological processes, such as adaptation to various environmental stresses, quorum sensing, bioflm formation, motility, and pathogenicity.The infection mechanism of E. piscicida is not to be fully understood. Especially, the regulatory roles of small non-coding RNA (sRNA) is almost unknown in the pathogenicity of bacterium. In this study, total RNA was extracted from E. piscicida grown in normal media (Con), acidic stress (Ac), iron defciency (Dp) and oxidation pressure (Pe) and subjected to RNA deep sequencing. A total of 148 sRNAs were found, including 19 previously annotated sRNAs and 129 novel sRNA. Compared in normal condition, the expression of 103 sRNAs showed signifcant diferences in three stress sample. Based on these results, sRNAs with differential expression under acidic and oxidative conditions (named sR082 and sR012) were selected for further study. Firstly, the expression levels of sR082 and sR012 in different growth phase of E. piscicida under various conditions were detected by qRT-PCR. The results showed that sR082 was highly expressed at early phase of bacterial growth and low pH conditions. And sR012 was highly expressed at logarithmic phase. To analyze the functions of sR082 and sR012, we constructed knockout strains TXΔsR082 and TXΔsR012 by In-frame deletion. The growth of knockout strains TXΔsR082 and TXΔsR012 and wild strains TXD1 was compared under low pH pressures and oxidative stress conditions. The results showed that the growth rate of TXΔsR082 significantly reduced under pH 5 conditions and the TXΔsR012 significantly decreased at entire growth phase compared with the wild-type strain under oxidative stress. These results indicated that sR082 and sR012 was related to the ability of bacteria to adapt to acid and oxidative stress environments, respectively.

To further analyze the effects of sR082 and sR012 on infection hosts, our study analyzed the virulence of TXΔsR082, TXΔsR012 and TXD1 for cell line, tissues and individuals, respectively. The results showed that compared with TXD1, the mutant strain TXΔsR082 and TXΔsR012 exhibited significantly reduction on infecting and replicating in flounder FG cells, dissemination in tissues, and lethality to fish. These observations suggest that sR082 and sR012 plays important roles in response to acid and oxidative stress and virulence of E. piscicida.

To further understand the role of sR012 in E. piscicida, we used TargetRNA2 to predict the sR012 target gene and performed qRT-PCR assay to detect the expression level of the putative target gene. The results showed that the total of four target genes (ETAE-0114, ETAE-2575, ETAE-3156 and ETAE-0911) were differentially expressed in TXΔsR012, and sR012 may regulate these four target genes to affect capacities of adaption oxidative stress and bacterial virulence.