文章摘要
克氏原螯虾细胞色素c基因通过调节凋亡途径抑制WSSV感染
Procambarus clarkii Cytochrome c Gene Inhibits WSSV Infection by Regulating Apoptosis Pathway
投稿时间:2021-08-27  修订日期:2021-09-27
DOI:
中文关键词: 克氏原螯虾  细胞色素c  凋亡  WSSV感染  RNA干扰
英文关键词: Procambarus clarkii  cytochrome c  apoptosis  WSSV infection  RNA interference
基金项目:江苏省自然科学基金面上项目(BK20181138);中央级公益性科研院所基本科研业务费专项项目(2019JBFZ09)
作者单位邮编
公洁 南京农业大学无锡渔业学院中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 214000
祝孟茹 南京农业大学无锡渔业学院中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 
占铭 南京农业大学无锡渔业学院中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 
席昌俊 南京农业大学无锡渔业学院中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 
神国卿 南京农业大学无锡渔业学院中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 
水燕 中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 
徐增洪 中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 
沈怀舜 南京农业大学无锡渔业学院中国水产科学研究院淡水研究中心淡水渔业与种质资源利用重点实验室 214000
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中文摘要:
      细胞凋亡是由一系列相关基因严格调控的细胞程序性死亡,在抵御病原入侵、维持机体内环境稳态等方面有着重要意义。其中细胞色素c从线粒体释放到细胞质中是凋亡开始的关键一步。本研究利用 RACE 技术首次克隆获得了克氏原螯虾(Procambarus clarkii)细胞色素c基因(PcCytc), 全长897 bp,包括163bp的5′-UTR、419bp的3′-UTR和315bp的开放阅读框,编码104个氨基酸。定量PCR检测结果表明, PcCytc基因在克氏原螯虾的各个组织中都有表达,其中在鳃,肠道和肌肉中表达高,在胃中表达最低。WSSV感染实验显示,在检测的肝胰腺、肠道和肌肉组织中PcCytc在病毒感染后表达水平均出现上调,并在24h达最高值,约是此时PBS组表达量的2.65、2.07和2.20倍,均存在极显著性差异(P<0.01)。PcCytc基因干扰后, 克氏原螯虾体内WSSV病毒拷贝数明显增加(P<0.05), 表明PcCytc能够抑制WSSV在克氏原螯虾体内的复制,延迟感染;同时凋亡相关基因bcl-2、bax、caspase-3的表达均发生显著上调或下调(P<0.05)。我们的结果表明PcCytc可通过调节凋亡途径抑制WSSV感染,论文结果为克氏原螯虾对WSSV感染的免疫反应提供了新的见解。
英文摘要:
      Apoptosis is the programmed cell death strictly regulated by a series of related genes. It is of great significance in resisting pathogen invasion and maintaining the homeostasis of the environment. Among them, the release of cytochrome c from mitochondria into the cytoplasm is a key step in the beginning of apoptosis. With the continuous in-depth study of Cytc in cell apoptosis and immunity, more and more evidences show that Cytc can participate in cell apoptosis induced by virus infection.For example, White Spot Sydrome Virus (WSSV) stimulation can induce Cytc gene expression in Litopenaeus vannamei hepatopancreas and hemocyte; the apoptosis of Epinephelus akaara hepatocytes induced by Red-spotted Grouper Nervous Necrosis Virus (RGNNV) is related to the release of Cytc. However, the role of Cytc-mediated apoptosis in Procambarus clarkii WSSV infection has not yet been reported. Therefore, in this study, the full length of the apoptosis-related gene PcCytc of P. clarkii was cloned, and the role of PcCytc in P. clarkii was analyzed. The expression in various tissues of P. clarkii proved that WSSV infection can induce the expression of PcCytc. In addition, the mechanism of PcCytc involved in cell apoptosis in the process of WSSV infection was also explored by RNA interference technology. In order to gain a deeper understanding of the potential role of apoptosis-related factors in the immune response of P. clarkii. In this study, the cytochrome c gene of P. clarkii (PcCytc) was cloned using RACE technology, with a total length of 897 bp, including 163 bp 5′-UTR, 419 bp 3′-UTR and 315 bp open reading frame. It encodes 104 amino acids. The structure prediction shows that PcCytc contains a conserved Cytochrom_C domain, which proves that it is related to energy production and tends to be conserved in evolution. The results of quantitative PCR showed that the PcCytc gene is expressed in all tissues of P. clarkii, among which the expression is the lowest in the stomach, and the expression is higher in the gills, intestines and muscles, which are respectively 9.46, 8.65 and 7.88 times of stomach expression. PcCytc shows relatively high expression in high energy consumption tissues such as intestines and muscles, which is consistent with previous studies in Penaeus vannamei. The highest expression level was observed in the gills of the main immune and respiratory tissues of P. clarkii, indicating that PcCytc may be involved in biological processes such as respiration and immunity. Based on the above results, we speculate that PcCytc may play different functions in different organizations. WSSV infection experiments showed that the expression level of PcCytc in the tested hepatopancreas, intestines and muscle tissues increased after virus infection, and reached the highest value at 24h (P<0.01), after which the expression level began to decrease until it returned to normal level at 96h, the overall performance is an induced expression pattern. It shows that PcCytc is involved in the process of WSSV infection. In addition, considering that PcCytc can participate in ATP production as a key element in the mitochondrial respiratory chain, the low expression of PcCytc leads to energy deficiency. We speculate that once the virus disrupts the energy metabolism of the host cell, the host may make up for the loss by up-regulating the expression of PcCytc. RNAi technology revealed the role of PcCytc in the process of WSSV infection. At 24 and 48 hours after WSSV infection, the WSSV copies of the PcCytc RNAi group was significantly increased compared to the uninterrupted group (P<0.01), and at 72h it was significantly increased (P<0.05). These results indicate that PcCytc plays an important role in inhibiting the replication of WSSV in P. clarkii and delaying the infection process. In order to further confirm whether PcCytc mainly inhibits WSSV infection through the apoptotic pathway, we tested the expression changes of some important apoptosis-related genes (bcl-2, bax, caspase-3). Among them, caspase-3 as an effector protein regulates cell apoptosis, and its expression directly reflects the result of cell apoptosis; the ratio of bcl-2/bax is considered to be an indicator of the process of cell apoptosis, and an increase in the ratio indicates that apoptosis has been affected. Inhibition, a decrease in the ratio indicates that apoptosis is promoted. The test results are as follows: Compared with the PBS group, the expression of bcl-2, bax and caspase-3 genes of P. clarkii in the WSSV group only was up-regulated to varying degrees, and there was a very significant difference (P<0.01). It shows that WSSV can cause hemolymph apoptosis in P. clarkii, which is consistent with the studies in mud crab, vannamei, and Chinese prawn. In addition, the expression of caspase-3 in the dsCytc injection group was significantly down-regulated (P<0.01), indicating that apoptosis was inhibited after interfering PcCyt; and the value of bcl-2/bax in the dsCytc injection group was significantly increased (P<0.01), which also proved this conclusion. In summary, our results indicate that PcCytc can inhibit WSSV infection by regulating the apoptotic pathway. The results of the paper provide new insights into the immune response of P. clarkii to WSSV infection.
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