白斑狗鱼LADD 2基因克隆及其组织表达特征分析  

Cloning and tissue expression characteristics analysis of LADD 2 gene in Esox lucius

作　　者：刘祎 王顺哲 刘璎慧 杨倩 汪泳昌 孙浩然 李胜忠[1] 张俊杰[1] LIU Yi;WANG Shun-zhe;LIU Ying-hui;YANG Qian;WANG Yong-chang;SUN Hao-ran;LI Sheng-zhong;ZHANG Jun-jie(Xinjiang Key Laboratory for Ecological Adaptation and Evolution of Extreme Environment Biology/College of Life Sciences,Xinjiang Agricultural University,Urumqi,Xinjiang 830000,China;College of Animal Science,Xinjiang Agricultural University,Urumqi,Xinjiang 830052,China;Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation,Ministry of Agriculture and Rural Affairs,Guangzhou,Guangdong 510380,China)

机构地区：[1]新疆极端环境生物生态适应与进化重点实验室/新疆农业大学生命科学学院,新疆乌鲁木齐830000 [2]新疆农业大学动物科学学院,新疆乌鲁木齐830052 [3]中国水产科学研究院珠江水产研究所/农业农村部热带亚热带水产资源利用与养殖重点实验室,广东广州510380

出　　处：《南方农业学报》2025年第1期305-313,共9页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32260915);新疆自然科学基金项目(2022D01A66)。

摘　　要：【目的】克隆白斑狗鱼(Esox lucius)LADD 2基因并分析其组织表达特征,为揭示LADD 2基因在白斑狗鱼性腺发育中的作用提供理论依据。【方法】利用白斑狗鱼性腺转录组数据,筛选得到126日龄雌性白斑狗鱼高表达基因LADD 2。克隆LADD 2基因编码区(CDS)序列,利用DNAMAN 9.0、NCBI ORF finder、BLAST、ClustalX、ExPASy、ProtParam、PSORT Ⅱ、SOMPA、SWISS-MODEL等在线软件进行生物信息学分析。基于鱼类梯型凝集素同源物及C型凝集素受体的氨基酸序列,使用MEGA 5.1中的邻接法(Neighbor-joining,NJ)构建系统发育进化树。采用实时荧光定量PCR检测LADD 2基因在126日龄、188日龄和320日龄白斑狗鱼性腺、肠道、肌肉、肝脏、鳃、脑、肾脏及头肾中的相对表达量。【结果】成功克隆获得白斑狗鱼LADD 2基因CDS序列,全长609 bp,编码85个氨基酸残基,蛋白分子量为9.78 kD,理论等电点(pI)为7.30,分子式为C_(8635)H_(13504)N_(2334)O_(2404)S_(115),为亲水性蛋白。LDDA 2蛋白中亮氨酸(11.86%)含量最高,色氨酸(2.28%)含量最低。蛋白磷酸化位点预测结果显示,LADD 2蛋白含有147个丝氨酸位点,98个苏氨酸位点,44个酪氨酸位点。蛋白结构预测结果显示,LADD 2蛋白主要由β-转角、α-螺旋和β-折叠构成,存在2个保守的PRT1结构域。氨基酸序列同源比对分析结果显示,白斑狗鱼与阿拉斯加黑鱼(Dallia pectoralis)LADD 2蛋白的相似性最高为99%。系统发育进化树显示,白斑狗鱼LADD 2蛋白与鱼类梯型凝集素同源物聚为一支,与大西洋鲑(Salmo salar)、银大麻哈鱼(Oncorhynchus kisutch)及鲱形白鲑(Coregonus clupeaformis)的亲缘关系较近。实时荧光定量PCR结果显示,随着发育时间延长,LADD 2基因在白斑狗鱼卵巢中的相对表达量逐渐降低,与精巢中LADD 2基因相对表达量差异越来越小,且在鳃和脑中也有一定表达。【结论】LADD 2基因在进化过程中具有一定的保守性和稳定性,其在白�【Objective】To clone LADD 2 gene of Esox lucius and analyze its tissue expression characteristics,which could provide theoretical basis for revealing the role of LADD 2 gene in the gonadal development of E.lucius.【Method】Using the gonadal transcriptome data of E.lucius,the highly expressed gene LADD 2 in 126-day-old female E.lucius was screened.The coding region(CDS)sequence of LADD 2 gene was cloned,and bioinformatics analysis was performed using online tools such as DNAMAN 9.0,NCBI ORF finder,BLAST,ClustalX,ExPASy,ProtParam,PSORT II,SOMPA and SWISS-MODEL.Based on the amino acid sequences of fish ladder-type lectin homologs and C-type lectin receptors,a phylogenetic tree was constructed using the neighbor-joining(NJ)method in MEGA 5.1.The relative expression levels of LADD 2 gene in the gonads,intestines,muscles,liver,gills,brain,kidneys and head kidneys of E.lucius at 126,188,and 320 day-old were detected using real-time fluorescence quantitative PCR.【Result】The CDS sequence of LADD 2 gene in E.lucius was successfully cloned,with a total length of 609 bp,encoding 85 amino acids residues.The protein molecular weight was 9.78 kD,the theoretical isoelectric point(pI)was 7.30,and the molecular formula was C_(8635)H_(13504)N_(2334)O_(2404)S_(115),indicating a hydrophilic protein.The LADD 2 protein had the highest content of leucine(11.86%),and the lowest content of tryptophan(2.28%).Phosphorylation site prediction showed that LADD 2 protein contained 147 serine sites,98 threonine sites and 44 tyrosine sites.Protein structure prediction revealed that LADD 2 protein mainly consisted of random coilsβ-turns,α-helices andβ-folds,with 2 conserved PRT1 domains.Amino acid sequence homology analysis showed that the similarity between the LADD 2 protein of E.lucius and that of Dallia pectoralis was the highest at 99%.The phylogenetic tree indicated that LADD 2 protein of E.lucius clustered with fish laddertype lectin homologs and was closely related to Salmo salar,Oncorhynchus kisutch and Coregonus clupeaformis.

关 键 词：白斑狗鱼 LADD 2基因 梯型凝集素 组织表达特征 

分 类 号：S965.199[农业科学—水产养殖]