Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi

C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990bp, containing a 71bp 5'-untranslated region (UTR), an open reading frame (ORF) of 723bp, and a 196bp long 3'-UTR. mC1qL2 cDNA was 1193bp, containing a 100bp 5'-UTR, followed by an ORF of 756bp and a 3'-UTR of 337bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed (p<0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection.     

The megalocytivirus-induced protein CsMig1 enhances Cynoglossus semilaevis resistance against viral infection

Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish species cultured in northern China. In this study, we identified and analyzed the expression and function of a megalocytivirus-induced gene, CsMig1, from tongue sole. The deduced amino acid sequence of CsMig1 is composed of 507 residues and contains no conserved domains. Blast analysis identified no close homologues of CsMig1. CsMig1 shares moderate sequence similarities in the N-terminal region with the Gig1 (i.e., grass carp hemorrhagic virus-induced gene) homologues of several teleost species. Quantitative real time RT-PCR analysis showed that constitutive CsMig1 expression occurred, in increasing order, in heart, spleen, muscle, kidney, liver, gill, and gut. Experimental infection with the viral pathogen megalocytivirus upregulated CsMig1 expression in kidney, spleen, and liver in time-dependent manners. Treatment of head kidney lymphocytes with the culture supernatant of megalocytivirus-stimulated cells significantly enhanced CsMig1 expression. When head kidney lymphocytes were transfected with the plasmid that constitutively expresses CsMig1, the cells exhibited significantly increased ability to resist megalocytivirus infection. Taken together, these results indicate that CsMig1 is a virus- and, possibly, interferon-induced novel immune factor that functions in the antiviral immunity of tongue sole.     

Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp

Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean‐fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059‐bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α‐helixes, eight β‐sheets, 16 regions of triple helical repeats, 21 low‐complexity regions, 10 function domains and two zinc‐binding sites; however, no calcium‐binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi‐quantitative RT‐PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real‐time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean‐fed grass carp.     

Molecular cloning and expression analysis of a CXC chemokine gene from large yellow croaker Pseudosciaena crocea

In the present study, we report the cloning of a CXCL12 chemokine gene homologue from the large yellow croaker Pseudosciaena crocea (LycCXCL12). The complete cDNA of LycCXCL12 is 678 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 11.1 kDa. The deduced LycCXCL12 contains a 22-aa signal peptide and a 75-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 57-68% and 32-36% aa sequence identities to known CXCL12 chemokines in fish species and other vertebrates, respectively. The LycCXCL12 gene was constitutively expressed in all tissues examined although at different levels. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL12 gene expression was significantly up-regulated in gills, liver, kidney, spleen and blood at 24 h after stimulation. Time course analysis using real-time PCR showed that LycCXCL12 gene expression reached peak level in spleen and kidney at 12 h or in gills at 24 h post-induction by poly(I:C), while its expression increased to the highest level in kidney at 24h or in gills and spleen at 48 h post-induction by bacterial vaccine, indicating that LycCXCL12 gene expression was differentially regulated by poly(I:C) and bacterial vaccine.     

Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) infection

The crustacean ectoparasite, Argulus poses one of the major threats to carp culture due to absence of any suitable control measure. The study was undertaken to determine the expression of immune-related genes in three major immunocompetent organs viz., kidney, skin and liver of rohu (Labeo rohita) during experimental freshwater lice Argulus siamensis infection. Results showed that the expression of TLR 22-like, lysozyme G and β2-microglobulin genes in kidney was significantly (P ≤ 0.05) down-regulated in lice-infected fish. On the other hand, no significant difference (P>0.05) in CXCa, lysozyme C, TNFα and complement component 3 (C3) expression was found between uninfected control and different degrees of lice infected fish. In the skin, the expression of TLR 22-like and TNFα genes were significantly up-regulated whereas that of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish with respect to control fish. The expression of CXCa, lysozyme C and transferrin was not detected in the skin samples of fish. In the liver, the expression of CXCa, lysozyme G, β2-microglobulin and transferrin was significantly (P≤0.05) up-regulated in lice-infected fish with respect to control fish whereas expression of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish. The expression of TLR 22-like, lysozyme C, TNFα genes was not detected in the liver samples of fish. This study indicates that majority of the genes showed down-regulation in kidney tissue whereas up-regulation in liver and skin tissues except C3 in Argulus-infected fish. We show that infection with this parasite irrespective of intensity can also result in immune gene expression changes in tissues situated away from the site of parasite attachment and feeding. The information obtained here could be useful towards understanding the susceptibility of rohu to argulosis and mechanisms involved in protection of rohu to ectoparasitic infections, which is causing immense economic losses to freshwater aquaculture sector.     

威宁绵羊SOCS7和GFAP基因组织表达研究

试验旨在检测细胞因子信号传导抑制蛋白7（suppressor of cytokine signaling 7，SOCS7）和胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）基因在威宁绵羊不同组织中的表达水平。以6月龄、1周岁和2周岁的威宁绵羊公、母羊为研究对象，采用实时荧光定量PCR测定威宁绵羊不同性别及不同生长阶段心脏、肝脏、脾脏、肺脏、肾脏及脑6个组织中SOCS7和GFAP基因的相对表达量，从mRNA水平上的探究两个基因之间的表达规律。结果显示，SOCS7基因在威宁绵羊不同组织中均有不同程度的表达，其中脾脏中相对表达量最高，其次是肺脏、肾脏、心脏、肝脏和脑组织，随着威宁绵羊年龄的增大，SOCS7基因在组织中的相对表达量呈现小幅度上升的趋势；GFAP基因在威宁绵羊脑组织中相对表达量最高，其余组织中表达量较低，随着年龄增长总体呈现下降趋势。从性别差异来看，威宁绵羊SOCS7基因相对表达量母羊普遍高于公羊，而GFAP基因则是公羊普遍高于母羊，推测SOCS7基因很有可能负向调控GFAP基因的表达。     

鸭β-防御素-2基因的克隆测序与组织表达分析

利用RT-PCR技术,从肝脏组织中扩增到鸭AvBD2基因.经测序表明,扩增到的鸭AvBD2大小为350 bp,含有1个大小为195 bp的开放阅读框,编码64个氨基酸残基.组织表达分析表明,鸭AvBD2基因在鸭脾脏和肾脏中大量表达;心脏和肝脏中有少量表达;肺脏和骨髓有中等水平表达;在胸腺、腔上囊、小肠、胰腺、腺胃、食管、气管、舌头、胸肌、卵巢、皮肤中未见表达.     

九龙牦牛PPARγ基因的克隆及其表达谱分析

根据普通牛(Bos tarus)过氧化物酶体增殖物激活受体γ(PPARγ)基因序列设计引物,用成年九龙牦牛(Bos grunniens)脂肪组织总RNA,经RT-PCR扩增获得了PPARγ基因序列(GenBank登陆号:GU061328),其中cDNA的ORF为1 428 bp,编码475个氨基酸,与普通牛PPARγ氨基酸的同源性达99%,有2个氨基酸发生突变。利用半定量RT-PCR分析九龙牦牛PPARγ基因的mRNA表达特性。结果表明:在脂肪、背最长肌、心、肝、肾、脾和肺脏中均检测到PPARγ基因的表达,并且在脂肪组织中表达量极显著高于其他组织(P0.01),在肝脏和脾脏中亦有较高表达。PPARγ基因在背最长肌中的表达5.5岁九龙牦牛显著高于0.5、3.5岁和9岁以上,其表达与背最长肌的肌内脂肪含量未见显著相关性。     

湘村黑猪sirt2基因多态性及其与肉质性状的关联分析和组织表达研究

本研究旨在探索沉默信号调节子家族(silent information regulator 1-7,SIRT1-7)sirt2基因在湘村黑猪不同组织间的表达及其多态性与肉质性状间的关联性,以期寻找与湘村黑猪肉质性状相关的分子标记。采用PCR-RFLP方法和基因测序技术对湘村黑猪sirt2基因多态位点进行分析,采用实时荧光定量PCR技术对sirt2基因在湘村黑猪心脏、肝脏、脾脏、肺脏、肾脏、胰腺、后腿肌和背最长肌8个组织中的相对表达量进行分析,利用SAS 9.4软件对sirt2基因突变位点不同基因型与肌肉色值、pH_(45 min)、pH_(24 h)、滴水损失、失水率、肌内脂肪、嫩度和眼肌面积进行关联分析。结果显示,在湘村黑猪sirt2基因第8外显子扩增片段中的240 bp处发现1处C→T碱基突变,编码氨基酸由精氨酸(Arg)变为半胱氨酸(Cys),为错义突变,并形成CC、CT和TT 3种基因型,CC基因型为优势基因型,C为优势等位基因,经χ~2检验表明该突变位点偏离哈代-温伯格平衡;该位点群体纯合度较高,有效等位基因数为1.301,多态信息含量为0.205,为低度多态(PIC<0.25)。基因多态性与肉质性状关联分析结果表明,TT基因型肉色L~*值和滴水损失显著低于CC和CT基因型(P<0.05),CC基因型失水率显著高于CT和TT基因型(P<0.05)。sirt2基因在湘村黑猪8个组织中均有表达,其中在背最长肌中相对表达量最高,与肺脏中表达量差异不显著(P>0.05),但显著高于心脏、肝脏、脾脏、肾脏、胰腺和后腿肌(P<0.05),且心脏、肝脏、脾脏、肾脏和胰腺中相对表达量差异均不显著(P>0.05)。本试验结果表明,sirt2基因对湘村黑猪肉质性状的发育有一定影响,可作为影响湘村黑猪肉质性状的候选基因进行深入研究。     

牛HSL基因的克隆和生物信息学分析

旨在克隆牛HSL基因的CDS序列,并对该基因进行生物信息学、组织表达谱分析。根据GenBank登录的牛HSL基因序列(NM_001080220)设计1对引物,对牛组织样品中的HSL基因CDS序列进行RT-PCR扩增及序列测定;利用半定量RT-PCR方法检测HSL基因在牛各组织中的表达情况;利用生物信息学软件对其所编码的牛HSL蛋白进行分析。结果显示,获得的牛HSL基因CDS全长为2271bp,编码756个氨基酸,与GenBank登录的牛HSL基因同源性最高,为99.9%。HSL蛋白含有一个HSL-Nsuperfamily保守结构域,无信号肽结构,具有疏水性。半定量RT-PCR显示,HSL基因在检测的心脏、脾脏、肺脏、肾脏、肝脏、大网膜、皮下脂肪、肌肉8种组织中均有表达,其中在大网膜和皮下脂肪中表达量较高,在肾脏、脾脏、肝脏、肺脏、肌肉和心脏中度表达。该试验可为研究牛HSL蛋白的结构和功能提供参考。     

金堂黑山羊白介素10基因的克隆及表达分析

为探究山羊白介素10（interleukin 10,IL-10）基因的序列特征及在组织中的相对表达情况,试验采用PCR法扩增并克隆金堂黑山羊IL-10基因,用DNAMAN、DNAStar、ExPASy等生物信息学软件进行序列分析,运用实时荧光定量PCR法检测IL-10基因在金堂黑山羊不同组织中的表达情况。结果显示,金堂黑山羊IL-10基因序列长为379 bp,开放阅读框为276 bp,编码91个氨基酸残基。IL-10蛋白二级结构中含有α-螺旋、β-转角和无规则卷曲,分别为76.92%、2.20%和20.88%。IL-10蛋白三级结构模型与人IL-10（1ilk.1.A）基因同源性最高（86.81%）。系统进化分析结果发现,金堂黑山羊IL-10基因与挪威山羊IL-10基因亲缘关系最近。实时荧光定量PCR检测发现,在金堂黑山羊肺脏中IL-10基因表达水平显著高于脾脏、心脏、肌肉和肾脏（P<0.05）,在肾脏中IL-10基因表达水平显著高于脾脏、心脏和肌肉（P<0.05）,在脾脏、心脏和肌肉中IL-10基因表达水平较低,但差异不显著（P>0.05）。本试验结果揭示了IL-10基因具有高度的保守性,可能参与调控山羊的免疫应答反应。     

STC-1基因在大、小体型猪中表达与分布的差异分析

试验分别采集40日龄小体型猪（巴马猪）和大体型猪（大白猪）的心脏、肝脏、脾脏、肺脏、肾脏、头骨、骨骼肌组织,利用实时荧光定量PCR检测斯钙素-1（stanniocalcin 1,STC-1）基因mRNA在各个组织中的表达水平,并通过Western blotting检测STC-1蛋白在各个组织中的分布。实时荧光定量PCR检测结果表明,STC-1基因mRNA在巴马猪和大白猪肺脏、肾脏中相对表达水平较高,在骨骼肌中的表达水平最低;除心脏和骨骼肌外,巴马猪其余各组织中STC-1基因mRNA表达水平均显著高于大白猪（P < 0.05）。Western blotting检测结果表明,巴马猪肝脏中STC-1蛋白的表达量最高,而大白猪脾脏中STC-1蛋白表达量最高,两者差异显著（P < 0.05）;巴马猪肺脏、肝脏、骨骼肌及心脏组织中STC-1蛋白表达量均极显著高于大白猪（P < 0.01）;而巴马猪肾脏、脾脏中STC-1蛋白表达量极显著低于大白猪（P < 0.01）。本研究首次对大、小体型猪不同组织的STC-1基因mRNA表达水平及其STC-1蛋白分布进行检测,导致该基因表达与分布差异的原因可能与两种猪受外界环境应激及生长发育差异有关。     

蛋氨酸对幼建鲤疾病抵抗能力及免疫应答的影响

本试验旨在研究蛋氨酸对幼建鲤疾病抵抗能力及免疫应答的影响.选择体重为(12.34±0.02)g健康建鲤864尾,平均分成6组,每组144尾(每组设3个重复,每个重复48尾),分别饲喂蛋氨酸水平为0.39%、0.70%、1.00%、1.30%、1.60%和1.90%的饲料,饲养60 d后用嗜水气单胞菌攻毒17 d,考察蛋氨酸对幼建鲤免疫功能的影响.结果表明:适宜蛋氨酸水平极显著提高了攻毒前头肾体指数、后肾体指数、脾体指数、血液红、白细胞数量和攻毒后红细胞数量(P<0.01);显著提高了攻毒前白细胞吞噬率和攻毒后成活率、白细胞数量、白细胞吞噬率、血清酸性磷酸酶活力、溶菌酶含量、补体C3含量、凝集素水平和抗嗜水气单胞菌抗体效价(P<0.05).由此得出,蛋氨酸通过增强白细胞吞噬能力和提高特异性抗体效价从而提高幼建鲤的非特异性和特异性免疫力,增强疾病抵抗力.     

Expression Pattern of HOXA1 Gene and Its Putative Regulating Mechanism of Yak

The aim of study was to obtain the yak HOXA1 gene sequence and related bioinformatics information,and analyze its expression spectrum and temporal expression profiles for providing related information of the formation of multiple vertebrae and its mechanism.RT-PCR technology was applied to clone the yak HOXA1 gene,semi-quantitative RT-PCR technique was used to detect the gene expression level in multiple organisms,and Real-time quantitative PCR technique was used to detect the gene expression level in the organisms in different development periods.The results showed that the obtained HOXA1 gene was 885 bp including the ORF of 870 bp,encoding 290 amino acids.The yak HOXA1 gene shared a homology of 75.4% to 98.1% with ordinary cow,wild yak,human,wild boar,horse,mice,chimpanzees,jungle fowl and wild boar.There were different expression level of HOXA1 gene in heart,liver,spleen,lung,kidney,large intestine,small intestine,muscle,stomach,ovaries,uterus,fallopian tubes,breast and testicular tissue.With the growth of the yak,HOXA1 gene expression level in tissue also increased.     

陆川猪GPR1基因真核表达载体构建及组织表达谱分析

试验旨在构建陆川猪G蛋白偶联受体1（G protein-coupled receptor 1，GPR1）基因真核表达载体，并对其组织表达谱进行分析。采用RT-PCR技术从10周龄陆川猪皮下脂肪组织中扩增出GPR1基因CDS区后，使用常规分子克隆手段构建含GPR1基因片段的真核表达载体pEGFP-N1-GPR1，利用双酶切和测序对重组质粒pEGFP-N1-GPR1进行鉴定，并以脂质体法将重组质粒转染3T3-L1细胞24 h后观察细胞荧光表达情况。收集所转染3T3-L1细胞并提取其总RNA，实时荧光定量PCR进一步检测GPR1真核表达载体表达情况；提取6头10周龄陆川猪心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、皮下脂肪总RNA，实时荧光定量PCR检测GPR1基因mRNA在陆川猪各组织中的表达量。结果表明，陆川猪GPR1基因CDS全长1 068 bp，成功将其连接至pEGFP-N1真核表达载体，重组表达载体pEGFP-N1-GPR1质粒和空载pEGFP-N1质粒所转染3T3-L1细胞均能表现出绿色荧光，且空白对照组并未表现出绿色荧光。实时荧光定量PCR结果证实，GPR1基因在重组质粒试验组的表达量极显著高于空载质粒组（P<0.01）。GPR1基因在10周龄陆川猪肝脏中表达量最高，在心脏、脾脏、肺脏、肾脏、皮下脂肪中均有表达，在背最长肌中几乎不表达。本试验成功构建了真核表达载体pEGFP-N1-GPR1，并获得了GPR1基因组织表达谱，为进一步研究GPR1基因对陆川猪脂肪沉积的影响提供参考。