Nucleotide polymorphism and function of piscine histone H2A in Edwardsiella piscicida infection
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摘要: 组蛋白是染色体核小体的重要组分，在调控染色质结构、基因转录、个体发育等不同生物学过程中起着重要作用。为了研究鱼类组蛋白基因是否存在核苷酸的多态性以及组蛋白核苷酸多态性是否会影响鱼类的抗病力，本实验以斑马鱼和草鱼为研究对象，通过PCR扩增克隆了组蛋白H2A的全长开放阅读框；利用过表达技术、菌落平板计数、感染存活分析以及荧光定量PCR技术，研究了斑马鱼和草鱼组蛋白H2A核苷酸多态性不同变异体在杀鱼爱德华氏菌感染中的作用。在本研究中，实验发现鱼类组蛋白H2A存在着丰富的核苷酸多态性。序列分析结果显示斑马鱼和草鱼组蛋白H2A核苷酸多态性的变异体核苷酸序列相似性为90%~100%；而两两H2A核苷酸多态性变异体氨基酸序列之间最多只有3个位点存在差异。通过体内和体外抗菌实验可知，斑马鱼和草鱼组蛋白H2A核苷酸的多态性显著影响H2A的抗菌活性。此外，筛选出的抗菌组蛋白H2A核苷酸多态性的变异体在斑马鱼体内的过表达，不仅具有免疫增强的作用，还能显著增强斑马鱼对杀鱼爱德华氏菌感染的抗病力。本研究为筛选具有抗病作用的组蛋白H2A免疫保护原奠定了重要基础。Abstract: Histones are central components of nucleosome and chromatin, which play critical roles in diversifying chromatin structure, transcriptional regulation, ontogenesis and so on. Although there were a lot of reports on the effects of piscine histones in the development, gene transcription regulation and anti-microbial properties of histone-derived antimicrobial peptides, the roles of histone nucleotide polymorphism in pathogen infection have not been reported in any species of vertebrates. In the present study, we found that nucleotide polymorphisms were abundant in zebrafish (Danio rerio) and grass carp (Ctenopharyngodon idella) H2A. There was 90%-100% identity among D. rerio and C. idella H2A variants at the nucleotide level. At most 3 sites of amino acid mutation existed between H2A variants from D. rerio or C. idella. The results from the in vitro and in vivo studies showed that the nucleotide polymorphism of D. rerio and C. idella H2A significantly affected the antibacterial activities of H2A. Furthermore, the overexpression of D. rerio antibacterial H2A variants in D. rerio embryos and/or larvae not only has the immune enhancement effect, but also enhances the resistence of D. rerio larvae against Edwardsiella piscicida infection. The present study established the immunological basis of histone H2A variants with nucleotide polymorphism in disease susceptibility or disease resistance.
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图 1 斑马鱼组蛋白H2A核苷酸多态性可变体核苷酸序列比较
Figure 1. Multiple nucleotide sequence alignments of D. rerio histone H2A variants with nucleotide polymorphism
图 2 斑马鱼组蛋白H2A核苷酸多态性可变体氨基酸序列比较
Figure 2. Multiple amino acid sequence alignments of D. rerio histone H2A variants with nucleotide polymorphism
图 3 草鱼组蛋白H2A核苷酸多态性可变体核苷酸序列比较
Figure 3. Multiple nucleotide sequence alignments of C. idella histone H2A variants with nucleotide polymorphism
图 4 草鱼组蛋白H2A核苷酸多态性可变体氨基酸序列比较
Figure 4. Multiple amino acid sequence alignments of C. idella histone H2A variants with nucleotide polymorphism
图 5 斑马鱼组蛋白H2A核苷酸多态性可变体体内抗菌作用
Figure 5. In vivo antibacterial activities of D. rerio histone H2A variants with nucleotide polymorphism
图 6 草鱼组蛋白H2A核苷酸多态性可变体体外抗菌作用
Figure 6. In vitro antibacterial activities of C. idella histone H2A variants with nucleotide polymorphism
图 7 zfH2A-5对抗菌模式识别受体(a)和抗菌蛋白(b)的表达调控
Figure 7. Regulation of zfH2A-5 on the expression of antibacterial PRRs (a) and antibacterial proteins (b)
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