克氏原螯虾自噬相关基因PcAtg2的克隆及其在白斑综合征病毒胁迫下的表达分析

Cloning and expression analysis of the autophagy related gene PcAtg2 in Procambarus clarkii under white spot syndrome virus stress

中文关键词: 克氏原螯虾  Atg2  基因克隆  WSSV  RNA干扰

英文关键词: Procambarus clarkii  PcAtg2  Gene cloning  White spot syndrome virus (WSSV)  RNA interference

中文摘要:

      为了解自噬相关基因Atg2在克氏原螯虾(Procambarus clarkia)先天免疫中的作用，本研究克隆了克氏原螯虾Atg2 (PcAtg2)基因全长序列。生物信息学分析显示，PcAtg2蛋白编码序列全长为9 966 bp，推测其编码2 189个氨基酸。组织定量表达分布显示，PcAtg2在克氏原螯虾的各个组织中均有表达，其中在肝胰腺中表达最高，在眼柄中表达最低。在白斑综合征病毒(WSSV)感染实验中，PcAtg2基因表达量在不同组织中均呈现显著上调趋势。RNA干扰(RNAi)实验显示，PcAtg2基因沉默后，WSSV在克氏原螯虾体内的增殖明显被抑制，同时，自噬相关基因的表达量上调。透射电镜分析结果显示，在WSSV感染后，PcAtg2基因沉默组中克氏原螯虾肝胰腺组织中的自噬小体多于对照组。本研究结果可为了解克氏原螯虾应对WSSV胁迫下的调控机制提供理论参考。

英文摘要:

      Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment. The incredible demand promotes the rapid development of the crayfish breeding industry. Viral diseases caused by white spot syndrome virus (WSSV) are widely spread in crustaceans, including P. clarkii. WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality. Virus infection can directly induce autophagy mechanisms. Autophagosomes can wrap virus particles and transport them to lysosomes for degradation. As a highly conserved cellular defense mechanism, autophagy plays an important role in the regulation of virus infections. However, many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication. In this study, WSSV in susceptible P. clarkii were explored to determine how autophagy related genes of P. clarkii participate in the regulation of virus infection. To study the role of the autophagy related gene (Atg2) in the innate immunity of P. clarkii, the full-length sequence of the Atg2 gene in P. clarkii (named PcAtg2) was cloned using the total RNA of P. clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique (RACE). The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P. clarkii was 9 966 bp, including a 582 bp 5' non coding region, 2 817 bp 3' non coding region, and 6 567 bp open reading frame. We speculate it encodes 2 189 amino acids. Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family, with 65 serine phosphorylation sites, and 48 glycosylation sites. The amino acid sequence of PcAtg2 in P. clarkii had the highest homology with the Homarus americanus Atg2 gene. The distribution of the PcAtg2 gene in the gill, heart, midgut, hepatopancreas, stomach, muscle, hemocyte, epidermis, testis, ovary, abdominal ganglion, and eyestalk of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that there was no significant difference in the expression of the PcAtg2 gene between male and female individuals. However, there were variations in expression in the different tissues. PcAtg2 was expressed in all tissues of P. clarkii, with the highest expression in the hepatopancreas and the lowest expression in the eyestalk. Under WSSV infection, PcAtg2 was initially up-regulated and then down-regulated in the different tissues, after induced expression. These findings suggest that PcAtg2 is involved in the regulation of autophagy in P. clarkii infected with the WSSV virus, and also plays an important regulatory role in the immune response. RNA interference (RNAi) technology was used to further explore the autophagy related genes PcAtg2 of P. clarkii and their role in WSSV infection. In the WSSV infection experiment with P. clarkii, the copy number of the WSSV virus in the dsPcAtg2 injection group was significantly lower than that in the control group and the dsGFP injection group, indicating that the replication of the WSSV virus was inhibited to some extent during the gene silencing of PcAtg2. The mortality results also showed that silencing PcAtg2 could reduce the mortality of P. clarkii infected with WSSV. In this experiment, after PcAtg2 was silenced, the transmission electron microscope images showed that after 24 and 48 hours of WSSV stress, autophagy vacuoles began to appear in the lysosomes in the hepatopancreas of P. clarkii in the control group, the injected dsPcAtg2 group, and the dsGFP injected group. More autophagosomes appeared and accumulated near the nucleus, indicating that P. clarkii can activate the regulation of cell autophagy under WSSV stress. Among them, more autophagosomes appeared in the hepatopancreas of P. clarkii in the dsPcAtg2 injection group, indicating the PcAtg2 gene promoted the formation of autophagosomes. WSSV virus proliferation can take advantage of autophagy. To avoid using the virus, cells will relatively down regulate the expression of autophagy related genes, and reduce the level of autophagy. In this experiment, by silencing the expression of the PcAtg2 gene, P. clarkii can promote autophagy regulation by up-regulating the expression of other autophagy related genes. In conclusion, the full-length sequences of the autophagy related gene PcAtg2 in P. clarkii were obtained for the first time, allowing us to reveal the effect and mechanism of WSSV infection on autophagy in P. clarkii. The effect of regulating autophagy on WSSV replication was analyzed, and the mechanism of the PcAtg2 gene acting on virus replication by regulating the formation of autophagosome was clarified. The PcAtg2 gene plays an important role in anti-virus immune defense in P. clarkii. We provide a theoretical basis for investigating anti-virus strategies from the perspective of autophagy. Further research on the host defense mechanism regulated by autophagy will provide new antiviral strategies.