Abstract Background Growth rate is a crucial economic trait for farmed animals, but the genetic regulation of this trait is largely unknown in non-model organisms such as shrimp. Results In this study, we performed genome-wide phenotypic quantitative trait loci (QTL) and expression quantitative trait loci (eQTL) mapping analyses to identify genes affecting the growth rate of Pacific white shrimp (Litopenaeus vannamei), which is the most commercially-farmed crustacean worldwide. We used RNA-sequencing of 268 individuals in a mapping population, and subsequently validated our findings through gene silencing and shrimp growth experiments. We constructed a high-density genetic linkage map comprising 5533 markers spanning 44 linkage groups, with a total distance of 6205.75 cM and an average marker interval of 1.12 cM. Our analyses identified 11 QTLs significantly correlated with growth rate, and 117,525 eQTLs. By integrating QTL and eQTL data, we identified a gene (metalloreductase STEAP4) highly associated with shrimp growth rate. RNA interference (RNAi) analysis and growth experiments confirmed that STEAP4 was significantly correlated with growth rate in L. vannamei. Conclusions Our results indicate that the comprehensive analysis of QTL and eQTL can effectively identify genes involved in complex animal traits. This is important for marker-assisted selection (MAS) of animals. Our work contributes to the development of shrimp breeding and available genetic resources.

为研究尼罗罗非鱼Rab11效应因子FIP5(Rab11-FIP5)的基因功能,试验设计了 Rab11-FIP5基因序列用于原核表达载体,构建原核表达载体pET-B2m-Rab11-FIP5,随后诱导表达融合蛋白Rab11-FIP5,表达产物纯化后免疫日本大耳兔制备Rab11-FIP5多克隆抗体,采用ELISA和Western Blot方法分析鉴定目的蛋白.结果:Western Blot检测结果显示有清晰目的条带,且条带单一,分子量约为62 KD,与预期分子量一致;ELISA检测抗体效价为1:2 048 000,说明效价符合抗体标准,性能良好.结论:成功制备了 Rab11-FIP5多克隆抗体,为开展Nt-Rab11-FIP5的定位和功能研究提供了基础工具.

INTRODUCTION:Golden pompano (Trachinotus ovatus) is economically significant important for offshore cage aquaculture in China and Southeast Asian countries. Lack of high-quality genomic data and accurate gene annotations greatly restricts its genetic breeding progress. OBJECTIVES:To decode the mechanisms of sex determination and rapid growth in golden pompano and facilitate the sex- and growth-aimed genetic breeding. METHODS:Genome assemblies of male and female golden pompano were generated using Illumina, PacBio, BioNano, genetic maps and Hi-C sequencing data. Genomic comparisons, whole genome re-sequencing of 202 F1 individuals, QTL mapping and gonadal transcriptomes were used to analyze the sex determining region, sex chromosome evolution, SNP loci, and growth candidate genes. Zebrafish model was used to investigate the functions of growth candidate gene. RESULTS:Female (644.45 Mb) and male (652.12 Mb) genomes of golden pompano were assembled and annotated at the chromosome level. Both genomes are highly conserved and no new or highly differentiated sex chromosomes occur. A 3.5 Mb sex determining region on LG15 was identified, where Hsd17b1, Micall2 and Lmx1a were putative candidates for sex determination. Three SNP loci significantly linked to growth were pinpointed, and a growth-linked gene gpsstr1 was identified by locus BSNP1369 (G → C, 17489695, Chr23). Loss of sstr1a (homologue of gpsstr1) in zebrafish caused growth retardation. CONCLUSION:This study provides insights into sex chromosome evolution, sex determination and rapid growth of golden pompano.

为研究卵形鲳鲹生肌调节因子(MRF)在胚胎发育中所发挥的重要作用,对卵形鲳鲹MRF基因家族进行了全基因组鉴定和生物信息学分析,并对其在13个胚胎发育阶段的基因表达进行了定量分析.结果表明:在卵形鲳鲹基因组中共鉴定出5个MRF基因家族成员:MyoD1、MyoD2、Myf5、Myf6和MyoG,分别编码297、263、240、231、250个氨基酸.MRF家族基因存在典型的BASIC与HLH结构域,其中MyoD1、MyoD2与MyoG分别定位于9号、1号与2号染色体,而Myf5与Myf6基因均定位于22号染色体且位于同一基因座.MRF家族基因胚胎定量表达研究表明,MyoG在受精卵到胚体形成期表达量极低,在眼囊、耳囊与心脏跳动期表达量迅速升高,而到晶体出现期表达量又有所降低,推测其可能在眼囊、耳囊与心脏跳动期发挥主要的生肌调节作用,而后期在维持肌肉形态或肌肉发育中仍发挥重要作用.MyoD1、MyoD2、Myf5与Myf6在受精卵到原肠中期表达量较低,而从原肠末期到晶体出现期表达量迅速升高(P<0.05),表明其主要从原肠末期开始发挥生肌调控作用.结果表明,MRF家族成员在胚胎发育过程中发挥重要的作用,但不同的MRF家族成员发挥作用的时间及功能可能不同.

[目的]制备斑马鱼zgc113227多克隆抗体和卵形鲳鲹EVM0008813多肽抗体,为后续深入研究斑马鱼zgc113227基因和卵形鲳鲹EVM0008813基因的功能特性提供重要工具.[方法]以卵形鲳鲹EVM0008813基因为查询序列,通过BLASTp搜索获得斑马鱼同源基因zgc113227(NP_001014341),以同源重组方式构建原核表达载体并转化大肠杆菌B21感受态细胞进行诱导表达,再以诱导表达获得的融合蛋白为抗原免疫日本大耳兔制备多克隆抗体,采用间接ELISA检测抗体效价,然后通过Western blotting鉴定及SDS-PAGE检测zgc113227多克隆抗体特异性,并以高效液相色谱—质谱联用(HPLC-MS)纯化鉴定EVM0008813多肽抗体.[结果]斑马鱼zgc113227和卵形鲳鲹EVM0008813均属于亲水性蛋白,二者的氨基酸序列相似性达58.12％,同时表现为潜在抗原位点多、柔性较强、表面可及性高,且无跨膜结构和信号肽存在.斑马鱼zgc113227和卵形鲳鲹EVM000881的保守结构域均包含5个保守基序(Motif 1～Motif 5),且发现1个相同的保守结构域(DDE_Tnp_4),故推测二者均属于DDE_Tnp_4家族.以zgc113227蛋白与等容积弗氏完全佐剂混合为抗原制备获得的zgc113227多克隆抗体效价为1:256000,特异性良好,可通过Protein G亲和层析柱进行纯化;以EVM0008813多肽与牛血清白蛋白(BSA)偶联为抗原制备获得的EVM0008813多肽抗体效价为1:256000,经HPLC-MS纯化鉴定抗体纯度达95.35％,特性良好.[结论]制备获得的斑马鱼zgc113227多克隆抗体和卵形鲳鲹EVM0008813多肽抗体纯度高、抗体效价高、特异性良好,可为斑马鱼zgc113227基因和卵形鲳鲹EVM0008813基因功能研究提供有利工具.

Background Holothuria scabra is a costly gourmet and traditional Chinese tonic medicine. However, the lack of high-quality genome information hinders the genetic, phylogenetic, and bioactivator researches. Objective To construct high-quality genomic data of H. scabra and conduct genome-wide phylogenetic analysis. Methods The whole genome of a male H. scabra was sequenced based on Nanopore MinION platform, and the sequence was assembled by wtdbg2. Transcriptome sequencing was used to aid the gene annotation. Repeat sequences, non-coding RNA, pseudogene and gene functional annotation were analyzed. 750 single-copy gene families from ten species were applied to construct phylogenetic tree for evolutionary analysis by using the ML method. Results The H. scabra genome of 1.18 Gb (N50 = 1557,492 bp) with 500.42 Mb of putative repetitive sequences was assembled from a male H. scabra individual, and 16,642 protein-coding genes, 951 pseudogenes, 1791 motifs and 45,400 domains from the generated assembly were identified. The divergence time between H. scabra and its ancestor was estimated approximately 192.6 million years ago. H. scabra and A. japonicas joined together while sea urchin and sea star diverged about 440 Mya ago. Some key genes involved in notochord and gill slits development, skeleton degeneration and nervous system, as well as homeobox genes differ between H. scabra and Apostichopus japonicas. Conclusion We report the first whole genome of H. scabra with expectation that this will be a valuable resource for genetic, phylogenetic, breading, molecular biology and bioactivator studies of sea cucumbers and other invertebrates.

[目的]明确卵形鲳鲹生长抑素(SST)家族基因的种类及其组织表达特征,为进一步揭示SST家族基因在其生长发育过程中的作用机制打下基础.[方法]采用HMMER 3.1对卵形鲳鲹全基因组数据进行搜索,然后通过Pfam、SMART及NCBI CDD等数据库确认搜索获得的基因是否属于SST家族基因;采用ProtParam和PSORT等在线软件进行生物信息学分析,并以实时荧光定量PCR检测SST家族基因在卵形鲳鲹不同组织中的表达情况.[结果]从卵形鲳鲹全基因组中共鉴定出4个SST家族基因(SST1、SST3、SST5和SST6),分别编码122、127、106和110个氨基酸残基,对应的编码蛋白分子量介于12249.3～14316.1 Da,理论等电点(pI)介于6.51～7.43,均定位于细胞外.4个卵形鲳鲹SST基因结构较简单且相似,均包含2个外显子和1个内含子;SST1和SST3基因位于7号染色体上,SST6和SST5基因则分别位于8号和23号染色体上;4个卵形鲳鲹SST氨基酸序列相似性的平均值为33.42％,以SST5氨基酸序列与SST6氨基酸序列的相似性最高(39.78％),SST3氨基酸序列与SST5氨基酸序列的相似性最低(28.16％).SST家族基因在卵形鲳鲹脑、胃、性腺和肌肉等组织中均有不同程度的表达,SST1、SST3和SST6基因在脑组织中显著高表达(P<0.05,下同),而SST5基因在性腺和肌肉组织中显著高表达;此外,SST3、SST5和SST6基因在卵形鲳鲹卵巢中的相对表达量均显著高于在精巢中的相对表达量.[结论]从卵形鲳鲹基因组中鉴定出4个SST家族基因(SST1、SST3、SST5和SST6),其表达分布存在组织特异性和种属特异性,可能介导卵形鲳鲹的神经调节、性腺发育及性二型性等多种生理功能,且不同物种的SST基因功能可能存在差异.

[目的]原核表达斑马鱼(Danio rerio)蛋白酪氨酸磷酸酶受体B(PTPRB)并制备多克隆抗体,为研究斑马鱼PTPRB基因功能及血管发育相关信号传导通路打下基础.[方法]采用无缝克隆技术将斑马鱼PTPRB基因插入原核表达载体pET-B2m构建重组表达质粒,转化大肠杆菌B21感受态细胞后采用IPTG进行诱导表达,然后以诱导表达的融合蛋白免疫大耳兔制备多克隆抗体,并采用Western blotting和ELISA检测多克隆抗体的特异性及免疫效价.[结果]斑马鱼PTPRB蛋白亲水性均值为-0.490,属于亲水性蛋白,且具有较丰富的潜在抗原表位位点,分布均匀,无典型的跨膜区.将斑马鱼PTPRB基因插入原核表达载体pET-B2m成功构建获得重组表达质粒pET-B2-PTPRB,转化B21感受态细胞后经IPTG诱导表达,即获得35.0 kD的融合蛋白.融合蛋白PTPRB主要以包涵体形式存在;以纯化融合蛋白PTPRB免疫大耳兔,其血清抗体效价为1:2048000,说明采用融合蛋白PTPRB可有效刺激大耳兔产生较强的免疫反应,获得较高效价的PTPRB多克隆抗体.Western blotting检测结果显示,PTPRB多克隆抗体具良好抗原特异性.采用Protein A/G亲和层析柱对制备获得的PTPRB多克隆抗体进行亲和层析纯化,可获得高纯度的多克隆抗体,纯化后的PTPRB多克隆抗体浓度在10 mg/mL以上.[结论]构建的斑马鱼PTPRB基因原核表达载体能高效表达具备良好免疫原性的融合蛋白PTPRB,以融合蛋白PTPRB免疫大耳兔可获得高效价、高特异性的PTPRB多克隆抗体,为研究斑马鱼PTPRB蛋白功能提供有利工具,也为揭示PTPRB在鱼类血管发育中的作用机制提供技术支持.

为研究肌生长抑制素(myostatin,MSTN)在卵形鲳鲹胚胎发育过程中的表达,对卵形鲳鲹MSTN基因CDS区进行克隆与分析,并对其14个胚胎发育阶段的表达量进行研究.结果显示:克隆获得MSTN基因1322 bp序列.该序列编码376个氨基酸,相对分子质量为42694.85 u,理论等电点pI为5.52,为带负电的蛋白.MSTN蛋白质不存在跨膜结构域,亚细胞定位主要于细胞外部,可能属于分泌蛋白质,易于表达和纯化.MSTN蛋白质无信号肽序列,预测含有17个磷酸化位点.对24种鱼类MSTN基因序列的同源分析发现:卵形鲳鲹MSTN与布氏鲳鲹同源性最高,与黄尾鰤同源性次之;3种鱼类均属鲹科Carangidae,而鲹科鱼类与锯盖鱼科Centropomidae鱼类遗传距离最小,具有较近的亲缘关系.MSTN基因在卵形鲳鲹从受精卵到晶体出现期的13个阶段均表达量极低或不表达,组间差异不显著(P>0.05),而在初孵仔鱼期表达量迅速升高,极显著高于前13个时期(P<0.01).MSTN基因可能在卵形鲳鲹孵化后发挥重要作用,而在孵化前作用微弱.

Precise regulation of angiogenesis is required for organ development, wound repair, and tumor progression. Here, we identified a novel gene, nxhl (New XingHuo light), that is conserved in vertebrates and that plays a crucial role in vascular integrity and angiogenesis. Bioinformatic analysis uncovered its essential roles in development based on co-expression with several key developmental genes. Knockdown of nxhl in zebrafish causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels and decreased sprouting in the caudal vein plexus. The nxhl gene also affects human endothelial cell behavior in vitro. We found that nxhl functions in part by targeting VE-PTP through interaction with NCL (nucleolin). Loss of ptprb (a VE-PTP ortholo) in zebrafish resulted in defects similar to nxhl knockdown. Moreover, nxhl deficiency attenuates tumor invasion and proteins (including VE-PTP and NCL) associated with angiogenesis and EMT. These findings illustrate that nxhl can regulate angiogenesis via a novel nxhl-NCL-VE-PTP axis, providing a new therapeutic target for modulating vascular formation and function, especially for cancer treatment.

One important goal of fish genetic breeding is to identify valuable loci and genes that can facilitate growth and thereby productivity. Few such loci or genes have been identified in golden pompano ( Trachinotus ovatus ), a species of significant economic value. In this study, we produced a high-quality chromosome-level genome assembly of the golden pompano by de novo sequencing and assemblies for 2 parents and 200 F 1 offspring by genome re-sequencing. We exploited these assemblies to identify loci and genes by QTL mapping, Kompetitive Allele Specific PCR (KASP) genotyping, and haplotype-based regional association analysis based on growth records of a 64 biparental and 147 individuals from a naturally occurring population. At a locus 291kb from BSNP21031, we identified a somatostatin receptor type 1-like (designated as gpsstr1) gene in which the BSNP1369 of the promoter region was highly associated with growth. Loss of sstr1a, the homolog of gpsstr1 in zebrafish, caused growth retardation. Sstr1a mediated growth via sstr2 and Wnt-gsk-3β signaling pathways. Our findings provide new insights into the underlying mechanisms controlling growth. Our strategy can serve as an effective way to uncover novel genomic information and facilitate improvement of fish growth.

为了解卵形鲳鲹(Trachinotus ovatus)SST1基因的生物学信息、胚胎发育不同时期的表达水平及其在组织中的表达和分布情况,进而探讨SST1基因可能的生理功能及作用机制.对卵形鲳鯵基因组(登录号:PRJNA574895)中的SST1基因(ID:EVM0001630)进行生物信息学分析,并对其在卵形鲳修胚胎发育的不同时期及其在幼鱼不同组织中的表达情况进行定量分析.结果 表明,卵形鲳鲹的SST1基因全长1155 bp,开放阅读框序列长1104 bp,共编码368个氨基酸,蛋白分子质量为41.42 ku,理论等电点pI为8.47,属于带正电的疏水性蛋白.SST1蛋白无信号肽序列,亚细胞定位主要于细胞质膜,属于膜蛋白,包含17个磷酸化位点,存在7个跨膜结构域,三维结构主体为7段α-螺旋并列扭曲排列成的一柱状结构.序列比对和系统进化树分析显示,卵形鲳鲹与黄尾鰤和高体鰤共聚于一个分支,氨基酸序列同一性分别为98.39％和98.37％.定量PCR分析结果表明,SST1在卵形鲳鲹胚胎发育的前13个时期微量表达,进入初孵仔期表达量迅速升高.SST1在卵形鲳鲹幼鱼不同组织中的表达量差异极显著,其中在脑组织中表达量最高,其次是卵巢和精巢,在背鳍、皮肤、肌肉等其他11个组织中的表达量相对较低.说明SST1在卵形鲳鯵神经内分泌、性腺发育及生殖细胞发生过程中可能发挥着重要作用.

[目的]明确卵形鲳鲹(Trachinotus ovatus)JunB基因(ToJunB)在胚胎发育过程中的表达规律,为揭示JunB基因在鱼类胚胎发育过程中的作用机制打下基础.[方法]采用RT-PCR扩增ToJunB基因编码区(CDS)序列,通过Prot-Param、NetSurfP 2.0、TMHMM、SignalP、PSORT及NetNGlyc 1.0等在线软件进行生物信息学分析,并采用荧光定量PCR检测ToJunB基因在卵形鲳鲹17个胚胎发育时期(受精卵、2-细胞期、8-细胞期、16-细胞期、32-细胞期、64-细胞期、多细胞期、高囊胚期、原肠早期、原肠中期、原肠末期、胚胎形成期、眼囊期、耳囊期、心脏跳动期、晶体出现期和初孵仔期)的表达情况.[结果]克隆获得的ToJunB基因(1777 bp)包含344 bp的5'端非编码区(5'-UTR)、954 bp的开放阅读框(ORF)及479 bp的3'端非编码区(3'-UTR),共编码318个氨基酸残基;其编码蛋白分子量为35.03 kD,理论等电点(pI)为8.26,呈碱性;总平均疏水指数(GRAVY)为-0.567,为亲水性蛋白.ToJunB蛋白无跨膜结构和信号肽,主要定位于细胞核,属于非分泌型蛋白,在第39、129和168位氨基酸处各存在1个潜在的糖基化位点;其二级结构中α-螺旋占35.67％、延伸链占14.33％、无规则卷曲占50.00％.基于JunB氨基酸序列相似性构建的系统发育进化树显示,卵形鲳鲹与黄尾鰤的遗传距离最近,均隶属于鲈形目鲹科.ToJunB基因在胚胎发育前期(受精卵至原肠早期)的相对表达量较高,至原肠中期达最高值,在胚胎发育后期(原肠末期至初孵仔期)的相对表达量均较低,且ToJunB基因在受精卵至原肠中期共10个发育时期的相对表达量极显著高于胚胎发育后期的7个时期(P<0.01).[结论]ToJunB基因在卵形鲳鲹胚胎受精卵至原肠早期的相对表达量较高,于原肠中期达最高值后迅速降低,原肠末期至初孵仔期的相对表达量均较低,说明JunB基因在卵形鲳鲹胚胎发育的细胞分裂和增殖过程中发挥重要作用.

为了研究尼罗罗非鱼(Oreochromis niloticus)蛋白激酶C theta (PKCθ)和Spartin的蛋白表达情况,本实验在大肠杆菌(Escherichia coli)中表达和提纯了尼罗罗非鱼PKCθ和Spartin的重组蛋白,并利用日本大耳兔(Oryctolagus cuniculus)制备了相应的多克隆抗体.用间接ELISA技术检测抗体效价,Western Blot鉴定抗体的特异性,并检测其在罗非鱼肝、脾、肠和肌肉组织中的表达情况.结果 表明,实验成功构建了原核表达载体pET-B2m-PKCθ和pET-B2m-Spartin,实现了重组蛋白的原核表达和纯化.PKCθ重组蛋白主要存在于包涵体中,分子量约为56 ku;Spartin重组蛋白分子量约为30 ku,以包涵体蛋白的形式存在.获得的多克隆抗体效价均高达1∶512 000,Western Blot检测结果表明,制备的抗体能特异性识别尼罗罗非鱼PKCθ和Spartin多种异构体;PKCθ蛋白在罗非鱼肝、脾、肠与肌肉组织中均有表达;Spartin蛋白在罗非鱼的肝、脾和肠组织中不表达,在肌肉组织中表达.研究表明,尼罗罗非鱼PKCθ和Spartin重组蛋白在大肠杆菌中成功表达,获得了高效价的多克隆抗体,并明确了尼罗罗非鱼PKCθ和Spartin在不同组织中的表达情况,为进一步研究尼罗罗非鱼PKCθ和Spartin的功能及其作用机制奠定了基础.

[目的]明确卵形鲳鲹胚胎发育中肌细胞生成素基因(MyoG)的表达规律,为阐明MyoG在卵形鲳鲹胚胎生长发育中的作用机制打下基础.[方法]采用RT-PCR扩增卵形鲳鲹MyoG基因全长序列,通过ProtParam、GOR IV、SWISS-MODEL、TMHMM、SignalP、PSORT及NetNGlyc 1.0等在线软件对其进行生物信息学分析;运用实时荧光定量PCR检测MyoG基因在卵形鲳鲹不同胚胎发育时期(受精卵、卵裂期、囊胚期、原肠胚期、胚胎形成期、眼囊期、耳囊期、心脏跳动期、晶体出现期和孵化期)的表达情况.[结果]从卵形鲳鲹胚胎组织中克隆获得的MyoG基因全长为1379 bp,编码233个氨基酸残基,其蛋白分子量为26059.19 Da,理论等电点(pI)为8.72;不稳定指数为75.23,即MyoG蛋白稳定性较差;总平均疏水指数(GRAVY)为-0.764,属于亲水性蛋白.卵形鲳鲹MyoG蛋白无跨膜结构,无信号肽序列,在第49位氨基酸处存在1个潜在的糖基化位点;卵形鲳鲹MyoG蛋白主要存在于细胞质和微体中,不属于分泌蛋白,其二级结构中以无规则卷曲占比最高(59.23％),其次为α-螺旋(21.46％)和延伸链(19.31％).MyoG基因相对表达量在卵形鲳鲹胚胎发育过程中呈明显的先升高后降低变化趋势.其中,在胚胎发育的前期(受精卵至胚体形成期)MyoG基因相对表达量极低,但从眼囊期开始其相对表达量迅速升高,至心脏跳动期达最高值;眼囊期、耳囊期和心脏跳动期3个时期的MyoG基因相对表达量均极显著高于胚胎发育前5个时期(P<0.01,下同);从晶体出现期开始,MyoG基因相对表达量极显著降低.[结论]MyoG基因除了在卵形鲳鲹眼囊期、耳囊期和心脏跳动期的分化形成过程中发挥重要作用外,在卵形鲳鲹胚胎器官分化形成后仍发挥着重要作用.

Background: The tilapia aquaculture industry is facing heavy economic losses due to Streptococcus agalactiae (S. agalactiae) infections. While progress has been made in past years, the lack of a high-quality tilapia genome and transcript annotations makes systematic and comprehensive exploration for a non-coding RNA regulatory network associated with the infection process unfeasible, and it stunts further research focused on disease defense and treatment. Herein, single molecular real time sequencing (SMRT-Seq) and RNA-seq data were utilized to generate a high-quality transcript annotation. In addition, Changes in mRNA and non-coding RNA expression were also analyzed during a S. agalactiae infection in tilapia. Findings: In total, 16.79 Gb of clean data were obtained by sequencing on six SMRT cells, with 712,294 inserts (326,645 full-length non-chimeric reads and 354,188 non-full-length reads). A total of 197,952 consensus transcripts were obtained. Additionally, 55,857 transcript sequences were acquired, with 12,297 previously annotated and 43,560 newly identified transcripts. To further examine the immune response in Oreochromis niloticus following a S. agalactiae infection, a total of 470.62 Gb of clean data was generated by sequencing a library containing 18 S. agalactiae infected tilapia samples. Of the identified genes, 9911 were newly exploited, of which 7102 were functional annotated. Furthermore, 7874 mRNAs, 1281 long non-coding RNAs (out of 21,860 long non-coding RNAs), and 61 circular RNAs (out of 1026 circular RNAs) were found to be differentially expressed during infection, with the 1026 circRNAs not previously identified in tilapia. Moreover, k-means clustering and WGCNA analyses revealed that the immune response of tilapia to a S. agalactiae infection can be divided into three stages: cytokines driven rapid immune response, energy metabolism promotion, and the production of lysosomes and phagosomes. During this response, the head kidney and spleen have synergistic effects, while maintaining independent characteristics. Finally, lncRNA-mRNA (trans and cis), lncRNA-miRNA-mRNA, and circRNA-miRNA-mRNA regulatory networks were constructed and revealed that non-coding RNA is involved in the regulation of immune-related genes. Conclusions: This study generated a greatly-improved transcript annotation for tilapia using long-read PacBio sequencing technology, and revealed the presence of a regulatory network comprised of non-coding RNAs in Nile tilapia infected with S. agalactiae.

文章测定并比较了雌、雄卵形鲳鲹（Trachinotus ovatus）肌肉概略营养成分、氨基酸和脂肪酸,对氨基酸和脂肪酸进行了营养评价。结果表明,雄性肌肉脂肪含量和亮度值显著高于雌性（P＜0.05）,水分含量和剪切力显著低于雌性（P＜0.05）,肌肉蛋白质、灰分含量和其他物理性状无显著差异（P＞0.05）。雌、雄鱼肌肉中均检测出17种氨基酸,其中雄性肌肉中苏氨酸等7种氨基酸、非必需氨基酸、半必需氨基酸、鲜味氨基酸含量以及氨基酸总量显著低于雌性（P＜0.05）,其他氨基酸无显著差异（P＞0.05）。依据氨基酸评分标准（AAS）和化学评分标准（CS）,雌、雄鱼肌肉的第一、第二限制氨基酸均为蛋氨酸和缬氨酸。雌性肌肉必需氨基酸指数高于雄性,其必需氨基酸的组成比例基本符合FAO/WHO标准。雌、雄鱼肌肉各种脂肪酸含量、单不饱和脂肪酸、多不饱和脂肪酸以及EPA+DHA均无显著差异（P＞0.05）,但雄性肌肉中Σn-6PUFA/Σn-3PUFA高于雌性。综合评价表明,雌性比雄性肌肉具有更高的食用价值。

[目的]通过高通量测序技术调研卵形鲳鲹基因组数据并开发SSR分子标记,为卵形鲳鲹全基因组测序与组装、种质资源保护利用及良种选育提供技术支撑.[方法]通过Illumina Hiseq 2500测序平台对卵形鲳鲹基因组进行调研,采用K-mer方法对基因组大小、杂合率、G+C含量及序列重复性等进行分析,从调研数据中分析SSR的分布特征,筛选出多态性SSR位点,并对卵形鲳鲹养殖群体进行遗传多样性分析.[结果]卵形鲳鲹基因组大小为642.68 Mb,杂合率为0.31％,重复序列比例为30.19％,G+C含量为41.45％,提示卵形鲳鲹基因组为简单基因组.基因组初步组装结果显示,Contig总长度为627.23 Mb,N50、N90分别为8.21和1.71 Mb;Scalffold总长度为628.19 Mb,N50、N90分别为10.19和2.04 Mb.从卵形鲳鲹基因组调研数据中共检测出190121条SSR序列,SSR序列分布密度为295.8条/Mb.在所有SSR序列中,以二核苷酸重复基元最多(115557条),占60.78％;其次是三核苷酸重复基元(54839条),占28.84％;六核苷酸重复基元最少(1172条),占0.62％.在二核苷酸重复基元中以TG和AC的重复数较多,分别占二核苷酸重复基元总数的22.99％和21.76％.从合成的50对SSR引物中筛选获得29对多态性SSR引物,采用这29对SSR引物对卵形鲳鲹群体进行遗传多样性分析,结果发现29个SSR位点共检测到98个等位基因,其有效等位基因数(Ne)为1.3998～3.9123(平均为2.6690),期望杂合度(He)为0.2856～0.7444(平均为0.5965),多态信息含量(PIC)为0.2647～0.6968(平均为0.5195);在29个SSR位点中,高度多态性位点(PIC>0.50)有15个,其余14个为中度多态性位点(0.25<PIC≤0.50),说明卵形鲳鲹群体遗传多样性较丰富.[结论]卵形鲳鲹基因组为简单基因组,利用基因组调研数据可实现SSR分子标记大规模开发,且新开发的SSR分子标记可用于卵形鲳鲹群体遗传多样性分析.

利用PCR-SSCP技术对吉富罗非鱼HSP70基因多态性进行检测,并采用最小二乘法分析多态性与罗非鱼Oreochromis niloticus耐寒性状的相关性.结果发现,依据HSP70基因序列设计的引物HP70-1、HP70-4、HP70-6不同耐寒能力样本中的电泳图具有带型多态性,且该基因多态性与其耐寒性状显著相关.据此推断HSP70基因是潜在的罗非鱼耐寒相关基因.

为体外表达尼罗罗非鱼髓样分化因子(MyD88)蛋白并制备抗该蛋白的多克隆抗体,采用无缝克隆技术将尼罗罗非鱼MyD88基因转入pET-B2m原核表达载体,并转入大肠杆菌B21中诱导表达,将表达的MyD88蛋白经纯化后免疫大耳兔制备多克隆抗体,采用Western blot和ELISA检测抗体的特异性和效价.试验结果表明,本研究成功构建了pET-B2m-M yD88原核表达载体,诱导表达获得了48.9 ku的重组蛋白,免疫大耳兔获得效价为1:2048000的抗尼罗罗非鱼M yD88多克隆抗血清.Western blot检测结果显示,该抗体能够特异性的识别原核表达的MyD88蛋白.原核表达体系的建立及多克隆抗体的制备为进一步研究M yD88蛋白在尼罗罗非鱼先天性免疫中的功能奠定了基础.