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凡纳滨对虾肠道黏膜屏障对黄曲霉毒素B1和副溶血弧菌E1的响应
齐灿灿
学位类型硕士
导师王雷
2017-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业水产养殖
关键词凡纳滨对虾 肠道黏膜屏障 黄曲霉毒素 副溶血弧菌
其他摘要    为探究凡纳滨对虾肠道黏膜的屏障作用,分别利用黄曲霉毒素B1(AFB1)和副溶血弧菌E1(VPE1)对凡纳滨对虾进行攻毒实验,从组织学及基因表达层面探究凡纳滨对虾肠道对毒素及弧菌的响应机制,以期为对虾的健康养殖、疾病防治提供理论依据。
    为探究AFB1对凡纳滨对虾肠道黏膜屏障的影响,以投喂含有15 mg/kg AFB1饲料的凡纳滨对虾作为实验组,不含AFB1饲料投喂的凡纳滨对虾作为对照组,分别于第2、4、8、12 d取肠道组织,采用实时荧光定量PCR检测mTOR信号通路中的真核细胞起始因子4E结合蛋白(eif4ebp),真核翻译起始因子1a(eif4e1a),真核翻译起始因子2(eif4e2)和核糖体s6蛋白激酶(p70s6k)基因,与免疫相关的转录因子DorsalRelish基因,酚氧化酶原(proPO)基因以及黏蛋白样围食膜因子(mucin-like PM)基因表达水平的变化,利用组织切片技术探究AFB1对肠道组织形态的影响。结果表明,AFB1的添加会对mTOR通路相关基因的表达产生影响,实验组对虾eif4ebp基因自第2 d起发生显著上调;eif4e2eif4e1a基因皆在第8 d和第12 d被显著抑制;p70s6k基因在第2 d和第4 d呈现下调趋势,并于第12 d回升至初始水平。AFB1同时也刺激了免疫系统的响应,实验组Dorsal基因和Relish基因均被显著诱导;proPO基因于第4 d和第8 d显著上调并于第12 d回落至初始水平;mucin-like PM基因在第2-8 d均显著上调。AFB1的添加也破坏了凡纳滨对虾肠道正常的组织形态,出现上皮细胞核肥大,边缘模糊,上皮细胞层部分脱落等现象。结果表明,AFB1严重影响凡纳滨对虾肠道黏膜屏障的功能,不仅对肠道黏膜造成了机械损伤,同时对肠道化学屏障和免疫屏障产生了影响。
    利用转录组测序(RNAseq)技术及生物信息学分析,探究了VPE1感染12 h时凡纳滨对虾肠道中与肠道黏膜屏障功能相关的基因转录水平的变化,结果表明:(1)转录组测序原始数据经过质控、拼接后,共得到164,420条Unigene,将Unigene与Nr,Nt等7大数据库进行比对,共有48542条Unigene注释到至少一个数据库中,占全部Unigene的29.52%;(2)利用DEseq进行差异表达分析,得到差异表达的基因(Differentially expressed genes,DEGs)共4646个,其中上调的基因有2469个,下调的基因有2177个;(3)从DEGs中鉴别出可能参与到凡纳滨对虾肠道黏膜屏障对VPE1感染响应过程的基因,涉及到肠道黏膜的机械屏障、化学屏障和免疫屏障的相关功能,包括:围食膜因子,细胞骨架和细胞连接,模式识别受体,抗菌肽与免疫信号通路,丝氨酸蛋白酶/蛋白酶抑制剂与酚氧化酶系统,细胞凋亡与细胞吞噬,抗氧化系统等功能。(4)利用实时荧光定量PCR(RT-qPCR)方法对15个DEGs进行验证,验证结果与转录组结果一致。
    利用组织切片和透射电镜观察VPE1浸浴后凡纳滨对虾肠道组织与细胞形态结构的变化。结果表明:(1)VPE1感染后,肠道上皮细胞层结构被破坏,上皮细胞层与基膜分离;(2)透射电镜结果表明,VPE1感染后,肠道上皮细胞的结构发生变化,细胞连接被破坏,细胞间界限模糊;部分细胞的细胞核固缩,异染色质聚集在核膜内围,溶酶体出现在细胞核附近;部分细胞微绒毛脱落,细菌进入细胞内部并被囊泡包裹,细胞内部出现空洞,有凋亡小体出现。同时电镜观察结果在一定程度上与RNAseq差异表达基因的结果相互印证。转录组、组织切片与电镜结果表明,机械、化学及免疫屏障参与到凡纳滨对虾肠道对VPE1感染的响应过程中,在凡纳滨对虾肠道抵御弧菌感染的过程中发挥重要作用。
    上述结果有助于理解凡纳滨对虾肠道对弧菌及毒素的响应机制,可为对虾肠道黏膜屏障研究提供理论依据,所获得的大量转录本可为未来对虾基因组学的研究提供参考,为新基因的发现提供丰富的数据资源。
; Litopenaeus vannamei were challenged by aflatoxin B1 and Vibrio parahaemolyticus E1 respectively, and changes of microstructure and ultrastructure as well as expression level were determined to investigate the mechanism of shrimp intestinal barrier responding to toxin and vibrio infection.

In order to determine the impact of Aflatoxin B1(AFB1) on the intestinal mucosa of Litopenaeus vannamei, shrimps were fed with normal diet and diet containing 15mg/kg of AFB1 for 12 days, and the tissues of intestine were collected at the 2rd, 4th, 8th and 12th day of the experiment. The relative expression of genes in mTOR signal pathway including eif4ebpeif4e1aeif4e2 and p70s6k, the immune-related genes including DorsalRelish and proPO and mucin-like PM gene were quantified with RT-qPCR. The changes of intestinal morphology were explored under microscope. The results showed that, the expression of genes in mTOR signal pathway were affected by AFB1, eif4ebp gene were up-regulated since the 2rd day, and decreased after reaching its peak at the 8th day; eif4e2 and eif4e1a gene were both inhibited at the 8th and 12th day; p70s6k gene were down-regulated from the 2rd to 8th day, and climbed to the initial level at the 12th day. The immune response of the intestine was also stimulated by AFB1, Dorsal and Relish gene were induced significantly after fed with AFB1; proPO gene was up-regulated at the 4th and 8th day, following by a fall to the initial level at the 12th day; and the mucin-like PM gene was also significantly induced at the 2th,4th and 8th day. The morphology of shrimp gut was changed by AFB1, the nuclear of epithelial cells swelled, the clear margin of chromatin was no longer present, and the ecclasis of epithelial cell layer occurred. In conclusion, the function of intestinal mucosal barrier was affected by AFB1, not only the mechanical barrier was damaged but also the chemical and immunologic barrier were impaired.

Transcriptome expression, microstructure and ultrastructure were analyzed to get a better understanding of the mechanism of shrimp intestinal barrier responding to vibrio infection. A total of 164420 unigenes were obtained from RNAseq data after quality control and assembly, and 29.52% of the unigenes were annotated to at least one of the 7 databases (Nr, Nt, GO, KOG, PAMF, SwissProt and KEGG). 4646 differentially expressed genes (DEGs) were determined by DEseq, and 2469 were upregulated while the other 2177 were downregulated. Genes related to intestinal barrier were determined involving in various functions of mechanical barrier, chemical barrier and immunologic barrier, including peritrophin, cytoskeleton and cell junction, pattern recognition receptors, antimicrobial peptide and immune signaling pathways, serine protease/ protease inhibitor and proPO system, apoptosis and phagocytosis, antioxidant and ROS system. Fifteen differentially expressed genes were selected for validation by RT-qPCR and the results were consistent with the RNA-seq data.

Microstructure and ultrastructure were significantly changed after VPE1 challenge. The epithelial layers detached from the basement membrane. The structure of cell junction was destroyed, nucleus pycnosis and secondary lysosome were also observed. Some of the cells lost their microvilli, cells containing bacterial cells and apoptotic bodies were occasionally seen. These results showed functions of mechanical barrier, chemical barrier and immunologic barrier involved in the response of intestinal barrier to the vibrio infection, and played significant roles in the defense of intestinal barrier. Our data contribute to improve the current understanding of host-Vibrio interaction, providing an abundant source for identification of novel genes.
学科领域水产学
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136604
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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齐灿灿. 凡纳滨对虾肠道黏膜屏障对黄曲霉毒素B1和副溶血弧菌E1的响应[D]. 北京. 中国科学院大学,2017.
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