Wang Lingling, Sun Xin, Zhou Zhi, Zhang Tao, Yi Qilin, Liu Rui, Wang Mengqiang, Song Linsheng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Dev Comp Immunol. 2014 Jul;45(1):123-32. doi: 10.1016/j.dci.2014.02.013. Epub 2014 Mar 2.
VP28 protein has been reported to work as a "vaccine" to protect the host from white spot syndrome, but the detailed mechanism of vaccination with VP28 protein in shrimp is still far from well understood. In the present study, whole transcriptomes of shrimp haemocytes were sequenced using the SOLiD4 platform after the successive VP28 stimulation. Eight single-end fragment libraries were constructed and sequenced in the four groups including the VP28-VP28, PBS-VP28, PBS-PBS and BLANK group, and there were 243,949,667 single-end reads with length of 50bp obtained totally, with 14,800 genes further identified. After reads mapping and transcript assembling, 1027, 1539, 1158, 1091 and 1300 genes in five differentially expressed gene lists were obtained in the comparison of VP28-VP28 versus PBS-VP28, VP28-VP28 versus PBS-PBS, VP28-VP28 versus BLANK, PBS-VP28 versus PBS-PBS and PBS-VP28 versus BLANK, respectively. There were 555 differentially expressed genes responsive to the single VP28 stimulation after grouping the PBS-VP28_BLANK and PBS-VP28_PBS-PBS gene lists, and 269 ones responsive to the successive VP28 stimulation after grouping the VP28-VP28_BLANK, VP28-VP28_PBS-PBS and VP28-VP28_PBS-VP28 gene lists. In the GO enrichment analysis of the genes responsive to the single VP28 stimulation, five immune-related GO terms were observed among 14 increased terms, which included defense response to bacterium, response to stimulus, disruption of cells of other organism, killing of cells of other organism and response to bacterium. It was worth noting that the GO terms, response to stimulus and response to stress, were the most common annotation ones which accounted 28.7% and 18.8% of the total differently expressed genes, respectively. For the genes responsive to the successive VP28 stimulation, terms including actin filament-based movement and myosin heavy chain binding were mostly enriched in the Biological Process and Molecular Function category, respectively. In the Cellular Component category, the enriched GO terms were myosin VII complex, myosin V complex, myosin VI complex and myosin II complex. Furthermore, the most abundant GO term was oxidation-reduction process, followed by single-organism transport, neurogenesis and translation for 214 genes only responsive to successive VP28 stimulation. These results collectively indicated that the successive VP28 stimulation could modulate cytoskeleton integration and redox to promote the phagocytosis activity of shrimp haemocytes, which might protect effectively for shrimp against WSSV infection.
据报道，VP28蛋白可作为一种“疫苗”保护宿主免受白斑综合征侵害，但虾类中VP28蛋白疫苗接种的详细机制仍远未完全清楚。在本研究中，在连续的VP28刺激后，使用SOLiD4平台对虾血细胞的全转录组进行测序。构建了8个单端片段文库，并在包括VP28-VP28、PBS-VP28、PBS-PBS和空白组的四组中进行测序，共获得243,949,667条长度为50bp的单端读数，进一步鉴定出14,800个基因。经过读数映射和转录本组装，在VP28-VP28与PBS-VP28、VP28-VP28与PBS-PBS、VP28-VP28与空白、PBS-VP28与PBS-PBS以及PBS-VP28与空白的比较中，分别在五个差异表达基因列表中获得了1027、1539、1158、1091和1300个基因。在对PBS-VP28_空白和PBS-VP28_PBS-PBS基因列表进行分组后，有555个差异表达基因对单次VP28刺激有反应，在对VP28-VP28_空白、VP28-VP28_PBS-PBS和VP28-VP28_PBS-VP28基因列表进行分组后，有269个差异表达基因对连续VP28刺激有反应。在对单次VP28刺激有反应的基因的GO富集分析中，在14个增加的术语中观察到5个与免疫相关的GO术语，包括对细菌的防御反应、对刺激的反应、对其他生物体细胞的破坏、对其他生物体细胞的杀伤以及对细菌的反应。值得注意的是，对刺激的反应和对应激的反应这两个GO术语是最常见的注释术语，分别占总差异表达基因的28.7%和18.8%。对于对连续VP28刺激有反应的基因，基于肌动蛋白丝的运动和肌球蛋白重链结合等术语分别在生物过程和分子功能类别中大多被富集。在细胞成分类别中，富集的GO术语是肌球蛋白VII复合体、肌球蛋白V复合体、肌球蛋白VI复合体和肌球蛋白II复合体。此外，最丰富的GO术语是氧化还原过程，其次是单生物体运输、神经发生和翻译，这些是仅对连续VP28刺激有反应的214个基因。这些结果共同表明，连续的VP28刺激可以调节细胞骨架整合和氧化还原，以促进虾血细胞的吞噬活性，这可能有效地保护虾免受WSSV感染。
