海藻希瓦氏菌感染对半滑舌鳎肠道菌群结构及相关功能基因表达的影响

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摘要：【目的】明確海藻希瓦氏菌（Shewanella algae）感染对半滑舌鳎（Cynoglossus semilaevis）肠道菌群结构及相关功能基因表达的影响，揭示肠道菌群和肠道组织相关功能基因在疾病发生及免疫应答过程中的作用机制。【方法】以致病性海藻希瓦氏菌人工感染半滑舌鳎后，采用16S rDNA高通量测序技术探究其肠道菌群组成结构的变化情况，并利用实时荧光定量PCR检测分析半滑舌鳎肠道组织中参与疾病发生和免疫应答相关功能基因的表达规律。【结果】共测序获得118657条有效序列，按97%的序列相似度聚类后得到6732个OTUs。Alpha多样性分析结果显示，Shannon指数和Chao1指数以感染前（CG）的健康半滑舌鳎最高，在感染后12 h（12hpi）最低;感染海藻希瓦氏菌前后半滑舌鳎肠道优势菌门无明显变化，但不同类群的相对丰度发生变化。在属水平上，Elizabethkingia、曼噬甲壳菌属（Chitinophaga）、Brevinema、苯基杆菌属（Phenylobacterium）、假单胞菌属（Pseudomonas）、乳杆菌属（Lactobacillus）、Marivita和雷尔氏菌属（Ralstonia）的相对丰度在CG半滑舌鳎肠道菌群组成中占比最高，希瓦氏菌属（Shewanella）、Petrimonas、Proteiniphilum和Aminobacterium的相对丰度在12hpi的占比最高，食酸菌属（Acidovorax）、芽孢杆菌属（Bacillus）和弧菌属（Vibrio）的相对丰度在感染后24 h（24hpi）的占比最高。半滑舌鳎肠道组织相关功能基因的表达变化表现为：果糖二磷酸醛缩酶A基因（ALDOA）的相对表达量在24hpi时显著高于CG（P<0.05，下同）;磷脂酶B1基因（PLB1）、热休克蛋白70 kD蛋白1A基因（HSPA1A）、组氨酸三聚体核苷结合蛋白1基因（HINT1）和γ谷氨酰转移酶1基因（GGT1）的相对表达量显著高于CG和24hpi;海藻糖酶基因（TREH）的相对表达量在12hpi时显著低于CG和24hpi。【结论】半滑舌鳎感染海藻希瓦氏菌后其肠道菌群多样性降低、菌群结构发生变化，肠道组织中免疫功能相关基因（HSPA1A和HINT1）及代谢功能相关酶类基因（ALDOA、PLB1、GGT1和TREH）呈差异表达，说明海藻希瓦氏菌感染引起半滑舌鳎肠道微生态紊乱，且肠道组织中免疫功能相关基因和代谢功能相关酶类基因分别参与机体的免疫应答及疾病发生过程。

关键词： 半滑舌鳎;海藻希瓦氏菌;肠道菌群;16S rDNA高通量测序;实时荧光定量PCR

中图分类号： S941.42                               文献标志码： A 文章编号：2095-1191（2019）10-2300-08

Effects of infection with Shewanella algae on the microbial communities and expression of related functional genes in the intestine of Cynoglossus semilaevis

ZHANG Yan-yu1， HAN Zhuo-ran1， SUN Jing-feng1*， LYU Ai-jun1，

HU Xiu-cai1， LIU Jun-feng2

（1College of Fisheries， Tianjin Agricultural University/Tianjin Key Lab of Aqua-Ecology and Aquaculture， Tianjin 300384; 2Tianjin Yushengtang Biotechnology Co.， Ltd.， Tianjin  300404， China）

Abstract：【Objective】The purpose of this study was to investigate the changes of intestinal microbial communities and expression of related functional genes in the intestine of Cynoglossus semilaevis after artificial infection with the pathogenic Shewanella algae， and reveal the role of intestinal microbial flora and related functional genes in the process of disease occurrence and intestinal immune response in the host. 【Method】After C. semilaevis were artificially infected with the pathogenic S. algae，16S rDNA high-throughput sequencing technique was used to study the changes of intestinal microbial communities， and real-time fluorescence quantitative PCR technology was used to study the expression pattern of functional genes involved in the process of disease occurrence and immune response in the intestinal tissues. 【Result】A total of 118657 effective tags were obtained and assigned to 6732 OTUs based on a 97% sequence similarity level. The results of alpha diversity analysis showed that the indexes of Shannon and Chao1 were the highest in the control group （CG）， and the lowest in the group of 12 h post-injection（12hpi）. At phylum level，although the identified dominant intestinal bacteria were consistent between the control and infection groups， the relative abundance of the bacterial taxa varied. At genus level， the relative abundances of Elizabethkingia， Chitinophaga， Brevinema， Phenylobacterium， Pseudomonas， Lactobacillus， Marivita， andalstonia were the highest in the intestinal microbiota of CG; the relative abundances of Shewanella， Petrimonas， Proteiniphilum， and Aminobacterium in 12hpi group were more than those in other groups. Acidovorax， Bacillus， and Vibrio had the highest relative abundance in the group of 24 h post-injection （24hpi）. The expression patterns of related functional genes in the intestine of C. semilaevis were as follows：the relative expression of fructose drphosphate aldolase A gene ALDOA in 24hpi group was significantly higher than that in CG（P<0.05， the same below）; the relative expression of phospholipase b1 gene（PLB1）， heat shock protein 70 kD protein 1A gene（HSPA1A），histidine trimer nucleoside binding protein 1 gene（HINT1） and gamma glutamyltransferase 1 gene（GGT1）in 12hpi group was significantly higher than those in CG and 24hpi groups; the relative expression of trehalase gene（TREH） in 12hpi group was significantly lower than those in CG and 24hpi groups.【Conclusion】The structure of the microflora changes with the diversity of intestinal microflora of C. semilaevis decreasing after infection with S. algae. The immune function related genes（HSPA1A and HINT1） and metabolism-related enzyme genes （ALDOA， PLB1，GGT1 and TREH） are diffe-rentially expressed. It indicates that infection with S. algae results in the disturbance of intestinal microecology of C. semilaevis， and the immune function related genes and metabolism-related enzyme genes are involved in the process of immune response against bacterial infection and disease occurrence.

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