Jiangsu Province Engineering Research Center for Aquatic Animals Breeding and Green Efficient Aquacultural Technology, College of Marine Science and Engineering, Nanjing Normal University, Nanjing, Jiangsu Province, 210023, China.
Jiangsu Province Engineering Research Center for Aquatic Animals Breeding and Green Efficient Aquacultural Technology, College of Marine Science and Engineering, Nanjing Normal University, Nanjing, Jiangsu Province, 210023, China.
Fish Shellfish Immunol. 2020 Apr;99:107-118. doi: 10.1016/j.fsi.2020.02.003. Epub 2020 Feb 5.
Nuclear factor κB (NF-κB) plays a key role in the innate immunity of invertebrates. Relish belongs to the NF-κB family. In insects, alternative splicing induces the sequence diversity of the Relish gene. However, information on the roles of various relish isoforms in crustacean innate immune response is limited. Here, two alternatively spliced Relish isoforms (designated as SPcRelish and LPcRelish) were identified from freshwater crayfish (Procambarus clarkii), and functional analysis was performed. The Relish gene has 25 exons and 24 introns. The long isoform LPcRelish is fully spliced, whereas the short isoform SPcRelish is alternatively spliced and contains exon 1-9 and a retention of intron 9. LPcRelish contains the Rel homology domain (RHD), the ig-like, plexins, transcription factors (IPT), and ankyrin-repeat (ANK) inhibitory domain. However, SPcRelish contains only the RHD and IPT domain, and does not have an ANK domain. The transcripts of SPcRelish and LPcRelish can be regulated by Vibrio parahaemolyticus. The intestinal immunological barrier and bacterial balance in the intestine play crucial roles in host health. In this study, we analyzed the connection between Relish isoforms and the transcripts of antimicrobial peptides (AMPs) in intestine. The transcripts of all the tested AMPs, except ALF-41125, were upregulated by V. parahaemolyticus. The knock down of the SPcRelish gene resulted in a significant decrease in the expression levels of ALF-7032, ALF-13162, and Crustin-42012 during V. parahaemolyticus invasion. The expression levels of four AMP genes (ALF-41125, ALF-42430, Crustin-41354, and Crustin-42993) were obviously increased in V. parahaemolyticus-challenged SPcRelish-silenced crayfish. ALF-7032, ALF-9228, ALF-13162, ALF-42430, Crustin-41354, Crustin-42012, and Crustin-42993 were evidently downregulated in V. parahaemolyticus-infected LPcRelish-silenced crayfish. Overall, generating the two Relish isoforms by alternative splicing may be an important mechanism of the host immune system to promote molecular diversity, which results in the functional diversity of the relish transcription factor.
核因子 κB（NF-κB）在无脊椎动物的先天免疫中发挥关键作用。Relish 属于 NF-κB 家族。在昆虫中，选择性剪接诱导了 Relish 基因的序列多样性。然而，关于各种 relish 同工型在甲壳动物先天免疫反应中的作用的信息有限。本研究从淡水小龙虾（Procambarus clarkii）中鉴定出两种选择性剪接的 Relish 同工型（命名为 SPcRelish 和 LPcRelish），并进行了功能分析。Relish 基因有 25 个外显子和 24 个内含子。长同工型 LPcRelish 完全剪接，而短同工型 SPcRelish 则是选择性剪接的，包含外显子 1-9 和内含子 9 的保留。LPcRelish 包含 Rel 同源结构域（RHD）、免疫球蛋白样、外泌体、转录因子（IPT）和锚蛋白重复（ANK）抑制结构域。然而，SPcRelish 仅包含 RHD 和 IPT 结构域，没有 ANK 结构域。SPcRelish 和 LPcRelish 的转录本可以被副溶血弧菌调控。肠道的免疫屏障和肠道内的细菌平衡对宿主健康起着至关重要的作用。在本研究中，我们分析了 Relish 同工型与肠道中抗菌肽（AMPs）转录本之间的联系。除了 ALF-41125 之外，所有测试的 AMPs 的转录本都被副溶血弧菌上调。SPcRelish 基因的敲低导致在副溶血弧菌入侵过程中 ALF-7032、ALF-13162 和 Crustin-42012 的表达水平显著降低。在副溶血弧菌挑战的 SPcRelish 沉默小龙虾中，四个 AMP 基因（ALF-41125、ALF-42430、Crustin-41354 和 Crustin-42993）的表达水平明显增加。在副溶血弧菌感染的 LPcRelish 沉默小龙虾中，ALF-7032、ALF-9228、ALF-13162、ALF-42430、Crustin-41354、Crustin-42012 和 Crustin-42993 的表达水平明显降低。总之，通过选择性剪接产生两种 Relish 同工型可能是宿主免疫系统促进分子多样性的一个重要机制，从而导致 relish 转录因子的功能多样性。
