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红螯螯虾鳃细胞与血细胞分子标记的鉴定
钟苏鸣,朱坤,杨丰,李钫
0
(集美大学水产学院,福建 厦门 361021;自然资源部第三海洋研究所、自然资源部海洋生物遗传资源重点实验室,福建 厦门 361005)
摘要:
cc甲壳动物的鳃中富集了大量血细胞,在循环血细胞稳态调节和免疫防御中发挥重要作用,因此获得区分鳃细胞和血细胞的分子标记将有助于研究鳃血细胞的功能。本研究采用SDS-PAGE电泳法比较了红螯螯虾(Cherax quadricarinatus)鳃细胞和血细胞的蛋白表达谱,获得3条组织特异性蛋白条带,并通过质谱技术鉴定了8个候选蛋白。实时荧光定量PCR(qRT-PCR)分析表明,空泡型-H+-ATP合酶C亚基(vacuolar-H+-ATPase subunit C, V-H+-ATPase)在鳃细胞中的转录水平是在血细胞中的53.3倍;而α-2-巨球蛋白(alpha-2-macroglobulin, α2M)和卵黄膜外层蛋白(vitelline membrane outer layer protein I-like protein, VMO-Ia)在血细胞中的转录水平分别是在鳃细胞中的10.3倍和10.9倍。Western-blot和免疫荧光分析表明,VV-H+-ATPase和VMOIa蛋白分别在鳃细胞和血细胞中特异性表达。综上,VV-H+-ATPase和VMO-Ia可以用作区分螯虾鳃细胞和血细胞的分子标记,可为其他十足目甲壳动物鳃血细胞的研究提供参考。
关键词:  海洋生物学  红螯螯虾    血细胞  分子标记
DOI:10.3969/J.ISSN.2095-4972.20230113001
基金项目:
Identification of molecular markers in gill cells and hemocytes of crayfish Cherax quadricarinatus
ZHONG Suming,ZHU Kun,YANG Feng,LI Fang
(Fisheries College, Jimei University, Xiamen 361021, China;Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, MNR, Xiamen 361005, China)
Abstract:
Being rich in a large number of hemocytes, the gills of crustacean play an important role in the regulation of hemocyte homeostasis and immune defense. Molecular markers to distinguish gill cells from hemocytes are required for the study of hemocytes in gill. In this study, we compared the protein profiles of gill cells and hemocytes of crayfish Cherax quadricarinatus using SDS-PAGE electrophoresis. Eight candidate proteins were identified from tissue-specific bands by LC-MS/MS. The expression of these candidate proteins in gill and hemocytes were analyzed by quantitative real-time fluorescence PCR (qRT-PCR). The transcription of vacuolar-H+-ATPase subunit C (V-H+-ATPase) in gill was 53.3 folds of that in hemocytes. Whereas, the transcription of alpha-2-macroglobulin (α2M) and vitelline membrane outer layer protein I-like protein (VMO-Ia) in hemocytes was 10.3 folds and 10.9 folds of that in gills, respectively. Western blot and immunofluorescence analysis confirmed that V-H+-ATPase and VMO-Ia proteins were specifically expressed in gill cells and hemocytes, respectively. Therefore, -H+-ATPase and VMO-Ia can be used as molecular markers to distinguish crayfish gill cells from hemocytes and may also provide a reference for the study of gill hemocytes in other decapod crustaceans.
Key words:  marine biology  Cherax quadricarinatus  gill  hemocyte  molecular marker

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