缢蛏Toll样受体家族基因全基因组水平鉴定及其在弧菌刺激下的表达分析

Genome-wide identification of Toll-like receptor family genes in Sinonovacula constricta and their expression in response to Vibrio parahemolyticus infection

关键词:  缢蛏  Toll样受体  弧菌刺激  表达分析

Key words:  Sinonovacula constricta  Toll-like receptors  Vibrio challenge  Expression analysis

摘要:

缢蛏(Sinonovacula constricta)是我国传统的四大海水养殖贝类之一，养殖过程中易受病原菌感染而造成大量死亡和经济损失。Toll样受体(Toll-like receptor, TLR)是目前研究最多的模式识别受体，在无脊椎动物的先天性免疫中发挥着重要作用。研究缢蛏TLR分子特征及其免疫功能对预防和控制病原菌感染至关重要。本研究从缢蛏基因组序列中鉴定出42个TLR基因(ScTLR)，其中，33个编码典型的TLR蛋白，其他9个为TLR样蛋白。根据蛋白结构域特点，将典型的TLR蛋白分为2大类4个亚类，一类为多半胱氨酸簇TLR (mccTLR)，包括1个P型TLR和7个sPP型TLR；另一类为单半胱氨酸簇TLR (sccTLR)，包括16个sP型TLR和9个Ls型TLR。与其他9种软体动物TLR基因的类型和数目比较结果显示，缢蛏基因组中未发现其他软体动物中存在的V型和Twin-TIR型TLR，起源古老的mccTLR类群在软体动物中没有大规模的扩张，而起源较晚的sccTLR类群却发生了大规模的扩张。荧光定量PCR分析显示，6种ScTLR在检测的7种组织中普遍表达，且在血细胞、鳃和肝胰腺组织中高表达。最后，副溶血弧菌(Vibrio parahemolyticus)胁迫12 h和48 h的缢蛏鳃和肝胰腺转录组数据分析显示，副溶血弧菌感染前后9个TLR基因在鳃或肝胰腺中的表达量发生显著变化，6个基因(ctg118.25、ctg118.26、ctg356.25、ctg774.6、ctg681.6和ctg1513.5)在感染12 h或48 h后的鳃组织中表达差异显著，前5个基因表达量上调，仅ctg1513.5表达量下调；3个基因(ctg467.9、ctg2496.3和ctg903.17)在感染12 h或48 h后的肝胰腺中表达差异显著，其中，ctg467.9和ctg2496.3表达量下调，ctg903.17表达量上调。综上所述，本研究结果将为深入探讨不同TLR基因在缢蛏天然免疫中发挥的作用提供研究基础。

Abstract:

Molluscs do not have adaptive immune cells and corresponding antibodies in their bodies. They mainly rely on the innate immune system to protect themselves from various pathogens and foreign substances to maintain normal life activities. Pattern recognition receptors (PRRs) first sense pathogen-associated molecular patterns (PAMPs) during the innate immune response, triggering specific signaling pathways and resisting pathogenic invasion. Toll-like receptors (TLRs) are widely studied PRRs and play important roles in the innate immunity of invertebrates. The first TLR was found in Drosophila, which activated the transcription factor NF-κB signaling pathway to guide early embryonic development. Later, it was proved to play an important role in the immunity of Drosophila. A typical TLR protein includes three protein domains, the extracellular domain containing two to 45 leucine-rich repeats (LRRs), the transmembrane domain, and the intracellular region containing a TIR (Toll/interleukin-1 receptor) domain. According to their structure variation in the LRR extracellular domain, TLRs can be divided into two types, namely single cysteine cluster TLR (sccTLR) and multiple cysteine cluster TLR (mccTLR). The razor clam Sinonovacula constricta is an economically important bivalve species and one of the four traditional mariculture mollusks in China. However, deterioration of the rearing environment and various bacterial and viral disease outbreaks have caused significant economic losses to the S. constricta industry. Therefore, a deep understanding of the immune defense mechanisms in S. constricta would help implement effective disease resistance strategies. In this study, we identified all TLR genes in S. constricta using whole genomic resources. Firstly, we identified the S. constricta proteins with both TIR domain (PF01582) and LRR domain (PF13855) using the InterProScan. Secondly, we further searched the whole genome DNA sequence using the TBLASTN program and the TIR-LRR protein sequences as query to identify missed TLR genes during genome annotation. The TLR protein domains identified were analyzed with the SMART software (http://smart.embl-heidelberg.de/). The TLR proteins with incomplete domains were further corrected with the FGENESH+ program on the Softberry website (http://www.softberry.com/). Finally, a total of 42 S. constricta TLR (ScTLR) genes were identified. Among them, 33 genes encode typical TLR proteins with three domains, and the remaining nine genes encode TLR-like proteins lacking some domains. The typical S. constricta TLR proteins were classified into two types and four subtypes based on the protein domain structure characteristics. The type mccTLR includes one P-TLR and seven sPP-TLRs, while the type sccTLR includes 16 sP-TLRs and nine Ls-TLRs. Furthermore, the type and number of TLR genes were compared among ten species from four classes of mollusks, including S. constricta. The results showed that two types of TLR genes, V-type and Twin-TIR TLR, identified in other mollusks were not found in the S. constricta genome. The number of TLR genes varied dramatically between different species, wherein the owl limpet (Lottia gigantea) and the common octopus (Octopus sinensis) possessed 16 and 17 TLR genes, respectively, while the American oyster (Crassostrea virginica) owned more than 130 TLR genes. Even in the same taxonomic genus, different species had a vastly different number of TLR genes, such as the Pacific oyster (Crassostrea gigas), which belongs to the same genus as the American oyster, and possessed 83 TLR genes, obviously less than the American oyster. Evolutionarily, the anciently originated mccTLR genes in mollusks did not expand in number, while the recently originated sccTLR genes largely expanded in number. The qRT-PCR tissue-specific expression analysis showed that six TLR genes randomly selected were expressed in seven tissues, including the hemolymph, gill, hepatopancreas, gonad, foot, mantle, and siphon, being highly expressed in the hemolymph, gill, and hepatopancreas. Finally, the razor clams were infected with Vibrio parahemolyticus, and the gill and hepatopancreas tissues were collected at 12 h and 24 h post-injection (hpi) for further transcriptome analysis. The results showed that nine TLR genes were differentially expressed in the gill or hepatopancreas before and after V. parahemolyticus injection. Six genes (ctg118.25, ctg118.26, ctg356.25, ctg774.6, ctg681.6, and ctg1513.5) were differentially expressed in gill at 12 hpi or 48 hpi, in which only ctg1513.5 was down-regulated and the other five were up-regulated. Three genes (ctg467.9, ctg2496.3, and ctg903.17) were differentially expressed in the hepatopancreas at 12 hpi or 48 hpi, wherein ctg467.9 and ctg2496.3 were down-regulated, and ctg903.17 was up-regulated. In summary, our findings will pave the way for investigating the functions of TLR genes in the innate immune response to different pathogens.