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| 中国对虾甘氨酸脱羧酶的基因克隆、表达及其与抗WSSV的关联分析 |
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史晓丽1,2, 张莹雪1,2, 孟宪红1,2, 孔杰1,2, 栾生1,2, 罗坤1,2, 曹宝祥1,2, 曹家旺1,2, 陈宝龙1,2
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1.农业农村部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛 266071
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| 摘要: |
| 本研究采用RACE技术克隆获得中国对虾(Fenneropenaeus chinensis)甘氨酸脱羧酶基因(FcGLDC)的全长cDNA及DNA序列，并对其进行生物信息学分析。结果显示，FcGLDC基因的cDNA全长为3481 bp，其中，ORF为2829 bp，5¢UTR长17 bp，3¢UTR长86 bp。完整的阅读框编码942个氨基酸，分子量为104.66 kDa，预测的理论等电点为6.51。FcGLDC基因DNA序列全长共4964 bp，包含12个外显子和11个内含子。同源性及系统进化分析显示，FcGLDC基因与节肢动物的GLDC基因聚为一类；氨基酸序列比对发现，FcGLDC基因的蛋白序列与节肢动物的相似度最高，与内华达白蚁(Zootermopsis nevadensis)、体虱(Pediculus humanus corporis)和白纹伊蚊(Aedes albopictus)的相似度分别为71%、68%和68%。在鳃、肝胰腺和肌肉中的荧光定量PCR结果显示，FcGLDC在肌肉中的相对表达量最高，鳃中最低。WSSV感染后，该基因在鳃、肝胰腺和肌肉中呈现出了不同的时空表达特点。使用直接测序法结合质谱法，在该基因内部发现4个SNP位点，但是各位点与抗WSSV性状均不相关(P>0.05)。本研究表明，FcGLDC基因在对虾感染WSSV后的应答反应中或起一定作用。 |
| 关键词: 中国对虾 甘氨酸脱羧酶 基因克隆 表达 关联分析 |
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| 基金项目:中国水产科学研究院黄海水产研究所基本科研业务费(20603022017001)、国家自然科学基金(41676148)、泰山学者良种工程项目和现代农业产业技术体系专项资金(CARS-48)共同资助 |
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| cDNA Cloning of the GLDC Gene in Fenneropenaeus chinensis and Its Expression and Functional Analyses after WSSV Infection |
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SHI Xiaoli1,2, ZHANG Yingxue1,2, MENG Xianhong1,2, KONG Jie1,2, LUAN Sheng1,2, LUO Kun1,2, CAO Baoxiang1,2, CAO Jiawang1,2, CHEN Baolong1,2
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1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071
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| Abstract: |
| Glycine decarboxylase is a key glycolytic enzyme that is involved in both the glycolytic and gluconeogenic pathways. In this study, the glycine decarboxylase gene from the Chinese shrimp Fenneropenaeus chinensis (FcGLDC) was cloned and sequenced for the first time. The full-length cDNA sequence of FcGLDC was 3481 bp long and contained a 17 bp 5¢-UTR, an 86 bp 3¢-UTR, and a 2829-bp open reading frame that encoded a 942 amino acid peptide with a calculated molecular mass of 104.66 kDa and theoretical isoelectric point of 6.51. The full-length FcGLDC DNA sequence was 4964 bp long and contained 12 exons and 11 introns. Multiple sequence alignment showed its high similarity with the glycine decarboxylase genes from other arthropod species: Zootermopsis nevadensis (71%), Pediculus humanus corporis (68%), and Aedes albopictus (68%). Phylogenetic analysis revealed that FcGLDC was in the same class with those from the other arthropods. The FcGLDC transcript showed the highest expression in muscles and the lowest expression in the gill. Stimulation of the shrimp with white spot syndrome virus (WSSV) resulted in changes in FcGLDC expression profiles in the hepatopancreas, gill, and muscles. In the gill, the transcript level was approximately 4.47-fold higher than that of the control group (P<0.05) at 24 h post injection (hpi). In the hepatopancreas, no significant difference was observed between the WSSV-challenged group and the control group (P>0.05). In the muscles, the transcript levels increased significantly, by approximately 19.98-fold at 24 hpi and 4.21-fold at 72 hpi, relative to the control group levels (P<0.05). Four single nucleotide polymorphisms were identified, using direct sequencing and time-of-flight mass spectrometry. Association analysis indicated that there was no significant association between the genotype and WSSV resistance (P>0.05). These results show that FcGLDC is inducible and may be involved in the shrimp’s immune response to pathogens. |
| Key words: Fenneropenaeus chinensis Glycine decarboxylase Gene cloning Expression Association analysis |