摘要
本试验旨在研究水体铅的慢性胁迫对日本沼虾硫氧还蛋白和热应激蛋白系统基因表达及肠道菌群的影响。将450尾均重为(0.10±0.02)g的日本沼虾随机分为3组,每组3个重复,每个重复50尾,进行为期60 d的慢性胁迫试验。水体铅的胁迫浓度分别为0(对照组)、13.13和26.26μg/L。试验结束后,采用实时荧光定量PCR分析肝胰腺中硫氧还蛋白(Trx)、硫氧还蛋白还原酶(TrxR)、热应激蛋白60(HSP60)、热应激同源蛋白70(HSC70)和热应激蛋白90(HSP90)mRNA相对表达量,同时通过16S rRNA高通量测序对日本沼虾肠道菌群组成及多样性进行分析。结果发现:1)慢性铅胁迫抑制TrxR和Trx mRNA表达,26.26μg/L组TrxR和Trx mRNA相对表达量显著低于对照组(P<0.05)。2)随着铅浓度的增加,HSP60和HSC70 mRNA相对表达量逐渐降低,26.26μg/L组HSP60和HSC 70 mRNA相对表达量显著低于对照组(P<0.05),而HSP 90 mRNA相对表达量呈先增加后降低的趋势,13.13μg/L组HSP90 mRNA相对表达量显著高于其余2组(P<0.05)。3)从门水平上分析日本沼虾所有组的肠道菌群,其主要以3个优势菌门为主,分别为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和软壁菌门(Tenericutes)。采用常规方差统计肠道菌群丰度发现,未分类气单胞菌种(Aeromonas_unclassified)的相对丰度在铅胁迫后显著下降(P<0.05)。采用线性判别分析效应值(LEfSe)方法分析肠道宏基因组,发现对照组与26.26μg/L组肠道菌群存在差异,对照组中肠杆菌属(Enterobacteriales)、肠杆菌科(Enterobacteriaceae)、肠杆菌目(Enterobacter)、柠檬杆菌属(Citrobacter)、疣微菌目(Verrucomicrobiae)、疣微菌纲(Verrucomicrobiales)、疣微菌科(Verrucomicrobiaceae)相对丰度较高,而26.26μg/L组中变形菌纲(Deltaproteobacteria)、黄杆菌纲(Flavobacteriia)、黄杆菌目(Flavobacteriaceae)、黄杆菌科(Flavobacteriales)、黄杆菌属(Flavobacterium)、拟杆菌门(Bacteroidetes)和井杆菌属(Phreatobacter)相对丰度较高。由此可见,慢性铅胁迫降低日本沼虾肝胰腺Trx和TrxR的转录水平,调控HSP60、HSC70和HSP 90 mRNA相对表达量,且日本沼虾肠道内存在不受慢性铅胁迫干扰的核心微生物门类,但高浓度的铅胁迫会产生丰度有显著性差异的菌群,维持正常代谢的菌群丰度减少,与降解污染物、调节机体免疫和抗氧化应激相关的菌群丰度增加。
The aim of this study was to investigate the effects of chronic lead exposure on expressions of thioredoxin and heat shock protein system genes and intestine microbiota in Macrobrachium nipponense.Juvenile M.nipponense with an average weight of(0.10±0.02)g were randomly divided into three groups in three replicates(50 tails/replicate)for chronic lead exposure trial.The concentration of lead in water were 0(control group),13.13 and 26.26μg/L and exposure time lasted for 60 days.After the exposure trial,quantitative real-time polymerase chain reaction(qRT-PCR)was used to analyze the mRNA expressions of thioredoxin(Trx),thioredoxin reductase(TrxR),heat shock protein 60(HSP60),heat shock cognate protein 70(HSC70)and heat shock protein 90(HSP90).Meanwhile,the composition and diversity of intestine microbiota were analyzed by 16s RNA high throughput sequencing.The results showed as follows 1)chronic lead exposure inhibited the mRNA expressions of TrxR and Trx.The mRNA relative expressions of TrxR and Trx in the 26.26μg/L group were significantly lower than those in the control group(P<0.05).2)With the increase of lead concentration,the mRNA relative expressions of HSP60 and HSC70 decreased gradually,and the mRNA relative expressions of HSP60 and HSC 70 in 26.26μg/L group were significantly lower than those in control group(P<0.05).But the mRNA relative expressions of HSP90 increased first and then decreased,and the HSP90 mRNA relative expressions in 13.13μg/L lead group was significantly higher than that in the other two groups(P<0.05).3)Analysis of intestinal bacteria of all processing groups of M.nipponense at phyla level,three dominant phyla,Proteobacteria,Firmicutes and Tenericutes were observed in the three groups at the phylum level in M.nipponense.It was found that the relative abundance of Aeromonas_unclassified significantly decreased after lead stress by the conventional variance analysis approach.Intestinal metagenome was analyzed by LEfSe approach,and it was found that there were differences in intestinal bacteria between the control group and the 26.26μg/L group.The results showed that the relative abundant of Enterobacteriales,Enterobacteriaceae,Enterobacter,Citrobacter,Verrucomicrobiae,Verrucomicrobiales,Verrucomicrobiaceae were higher in the control group,and the relative abundant of Deltaproteobacteria,Flavobacteriia,Flavobacteriales,Flavobacteriaceae,Flavobacterium,Bacteroidetes,Phreatobacter were higher in the 26.26μg/L group.Thus it can be concluded that chronic lead exposure decreases the transcription level of Trx and TrxR genes and regulates the mRNA relative expressions of HSP60,HSC 70 and HSP 90 in the hepatopancreas of M.nipponense.Regardless of the concentration of the lead exposure,there are core microorganisms in the intestinal tract of M.nipponense,but high-concentration lead exposure will produce bacteria with significant difference in abundance,and the abundance of bacteria to maintain normal metabolism will be reduced,and the bacteria which is related to the degradation of pollutants and the regulation of immune and antioxidant stress will increase.
作者
郑纯纯
李小雨
聂欢
周厚杰
丁志丽
田钰滢
杜明川
刘翥
ZHENG Chunchun;LI Xiaoyu;NIE Huan;ZHOU Houjie;DING Zhili;TIAN Yuying;DU Mingchuan;LIU Zhu(Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and Development,Key Laboratory of Aquatic Animal Genetic Breeding and Nutrition of Chinese Academy of Fishery Sciences,College of Life Science,Huzhou University,Huzhou 313000,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2019年第11期5378-5390,共13页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金项目(31402308)
浙江省自然科学基金项目(LQ14C190004,LY16C190006)
关键词
热应激蛋白
硫氧还蛋白
肠道菌群
铅
日本沼虾
heat shock protein
thioredoxin
intestinal microbiota
lead
Macrobrachium nipponense
