Cis-eQTL Analysis and Functional Validation of Candidate Genes for Carcass Yield Traits in Beef Cattle

Carcass yield traits are of considerable economic importance for farm animals, which act as a major contributor to the world’s food supply. Genome-wide association studies (GWASs) have identified many genetic variants associated with carcass yield traits in beef cattle. However, their functions are not effectively illustrated. In this study, we performed an integrative analysis of gene-based GWAS with expression quantitative trait locus (eQTL) analysis to detect candidate genes for carcass yield traits and validate their effects on bovine skeletal muscle satellite cells (BSCs). The gene-based GWAS and cis-eQTL analysis revealed 1780 GWAS and 1538 cis-expression genes. Among them, we identified 153 shared genes that may play important roles in carcass yield traits. Notably, the identified cis-eQTLs of PON3 and PRIM2 were significantly (p < 0.001) enriched in previous GWAS loci for carcass traits. Furthermore, overexpression of PON3 and PRIM2 promoted the BSCs’ proliferation, increased the expression of MYOD and downregulated the expression of MYOG, which indicated that these genes may inhibit myogenic differentiation. In contrast, PON3 and PRIM2 were significantly downregulated during the differentiation of BSCs. These findings suggested that PON3 and PRIM2 may promote the proliferation of BSCs and inhibit them in the pre-differentiation stage. Our results further contribute to the understanding of the molecular mechanisms of carcass yield traits in beef cattle.     

2基因片段的克隆和反义表达载体的构建及遗传转化初步研究

以我国重要的生物能源灌木——中间锦鸡儿枝叶和种子为材料,根据Genbank中已经发表fad2基因的同源序列,利用PCR技术克隆得到两个基因片段.在GenBank中Blast(GenBank登录号AY957393和AY957394),所得基因片段和同属豆科的Glycine max Gm fad2-2a,同源性高达88 %,属于fad2基因编码区中间片断.将所得片段经BamHI和SacI酶切后插入表达载体质粒pBI121,构建了反义表达载体pBI121 fad2,并利用农杆菌介导法转入烟草叶片,获得了抗卡那霉素和安苄青霉素的再生烟草植株.初步分析结果表明,与对照烟草相比较,转基因烟草种子脂肪酸含量没有明显差异,而亚油酸则减少了10.3 %.这为进行下一步柠条分子改良,获得高单不饱和脂肪酸的优质中间锦鸡儿新品种以及改造其它木本生物柴油用木本原料植物奠定了基础.     

人类博卡病毒结构蛋白基因VP2的克隆与表达

采用PCR方法扩增出人类博卡病毒结构蛋白基因VP2,通过双酶切、连接、转化等方法将VP2基因克隆到原核表达载体pMAL-c2x上,构建重组质粒pMAL-c2x-VP2,通过双酶切检测重组质粒构建成功;用0.8 mmol.L-1IPTG诱导融合蛋白表达,经SDS-PAGE检测、Western Blot分析,证明目的蛋白得到了表达。可为目的蛋白的纯化及结构和功能的研究、相应抗体的制备打下坚实的基础。     

牛分枝杆菌mpb64-ag85b-esat-6融合基因真核表达载体的构建及其在SP2/0细胞中的表达

目的构建牛分枝杆菌mpb64-ag85b-esat-6融合基因真核表达载体,并在SP2/0细胞中表达。方法利用PCR技术和重叠延伸剪接(SOE)技术扩增牛分枝杆菌ag85b、mpb64、esat-6基因和mpb64-ag85b融合基因,克隆至真核表达载体pcDNA3.1(+)上,构建重组质粒pcMAE。将该重组质粒体外转染SP2/0细胞,间接免疫荧光法检测目的基因的表达。结果真核表达质粒pcMAE经酶切鉴定及测序,证实构建正确,转染后SP2/0细胞膜上出现均质的蓝绿色荧光。结论已成功构建了牛分枝杆菌mpb64-ag85b-esat-6融合基因真核表达载体,并在SP2/0细胞中获得表达,为进一步研制牛结核病多基因融合DNA疫苗奠定了基础。     

Evaluation of the Effect of the Fibroblast Growth Factor Type 2 (FGF-2) Administration on Placental Gene Expression in a Murine Model of Preeclampsia Induced by L-NAME

The abnormal implantation of the trophoblast during the first trimester of pregnancy precedes the appearance of the clinical manifestations of preeclampsia (PE), which is a hypertensive disorder of pregnancy. In a previous study, which was carried out in a murine model of PE that was induced by NG-nitro-L-arginine methyl ester (L-NAME), we observed that the intravenous administration of fibroblast growth factor 2 (FGF2) had a hypotensive effect, improved the placental weight gain and attenuated the fetal growth restriction, and the morphological findings that were induced by L-NAME in the evaluated tissues were less severe. In this study, we aimed to determine the effect of FGF2 administration on the placental gene expression of the vascular endothelial growth factor (VEGFA), VEGF receptor 2 (VEGFR2), placental growth factor, endoglin (ENG), superoxide dismutase 1 (SOD1), catalase (CAT), thioredoxin (TXN), tumor protein P53 (P53), BCL2 apoptosis regulator, Fas cell surface death receptor (FAS), and caspase 3, in a Sprague Dawley rat PE model, which was induced by L-NAME. The gene expression was determined by a real-time polymerase chain reaction using SYBR green. Taking the vehicle or the L-NAME group as a reference, there was an under expression of placental VEGFA, VEGFR2, ENG, P53, FAS, SOD1, CAT, and TXN genes in the group of L-NAME + FGF2 (p < 0.05). The administration of FGF2 in the murine PE-like model that was induced by L-NAME reduced the effects that were generated by proteinuria and the increased BP, as well as the response of the expression of genes that participate in angiogenesis, apoptosis, and OS. These results have generated valuable information regarding the identification of molecular targets for PE and provide new insights for understanding PE pathogenesis.     

人NIRF基因真核表达质粒的构建及其在HepG2.2.15细胞中的表达

目的构建人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达NIRF蛋白。方法从HeLa细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增NIRF基因编码区全长序列,插入到pIRES2-EGFP真核表达载体中,构建重组真核表达质粒pIRES2-EGFP-NIRF,转染HepG2.2.15细胞,检测细胞中NIRF基因mRNA的转录水平及蛋白的表达水平。结果重组真核表达质粒pIRES2-EGFP-NIRF经双酶切及测序鉴定证明构建正确,转染HepG2.2.15细胞后,可检测到细胞中NIRF基因mRNA的转录及蛋白的表达。结论已成功构建了人NIRF基因真核表达质粒,并在HepG2.2.15细胞中表达了NIRF蛋白,为下一步研究其在肿瘤组织中的功能奠定了基础。     

Single Nucleotide Polymorphisms in Growth Hormone Gene and Their Association with Growth Traits in Siniperca chuatsi (Basilewsky)

Growth hormone (GH) has been considered as a candidate gene for growth traits in fish. In this study, polymorphisms of the GH gene were evaluated for associations with growth traits in 282 Siniperca chuatsi individuals. Using directly sequencing, four single nucleotide polymorphisms (SNPs) were identified in GH gene, with two mutations in intron 4 (g.4940A>C, g.4948A>T), one mutation in exon 5 (g.5045T>C) and one in intron 5 (g.5234T>G). Notably, three of them were significantly associated with growth performance, particularly for g.4940A>C which was highly correlated with all the four growth traits. In conclusion, our results demonstrated that these SNPs in GH gene could influence growth performance of S.chuatsi and could be used for marker-assisted selection (MAS) in this species.     

argE基因的表达优化及Zn2+添加时间和浓度的影响机制分析

确定了目的基因argE在重组菌BL21(DE3)-pET22b-argE中的表达位置,研究了Zn2+对重组菌生长及表达产物活性的影响,并分析了影响机制. 结果表明,argE可在重组菌中高效表达,表达产物N-乙酰鸟氨酸脱酰基酶大多以不可溶的包涵体形式存在,只有少量为有活性的可溶性表达. 1.0 g/L的Mg2+对重组菌的生长及酶活有明显促进作用. Zn2+加入时机及加入量不同,影响结果也不同. 发酵起始加入Zn2+严重抑制菌的生长及酶活,而在1.0%乳糖诱导2.5 h后加入则可解除生长抑制并提高酶活. SDS-PAGE电泳及活力测定证实Zn2+参与形成酶的催化中心,对酶的表达量没有影响.     

汉坦病毒SEO型S基因的克隆及在大肠杆菌中的高效表达

目的构建汉坦病毒SEO型S基因原核表达载体。方法应用RT-PCR方法扩增汉坦病毒SEO型的YZG-Changchun株S基因,克隆至pMD18-T载体中,经酶切鉴定及PCR分析后,定向克隆入原核表达载体pET-28a中,转化E.coliRosetta,经IPTG诱导表达,SDS-PAGE和Western blot分析外源蛋白的表达情况。结果表达载体经双酶切和测序证明构建正确。IPTG浓度为1.0 mmol/L,诱导4.5 h时,S基因在原核细胞中得到了高效表达,表达的蛋白占菌体蛋白总量的37%,纯化后的蛋白具有良好的抗原活性。结论已成功构建了汉坦病毒SEO型S基因原核表达载体,并得到高效表达。     

JTV1基因真核表达质粒的构建及其在K562细胞中的表达

目的 构建JTV1基因真核表达质粒并稳定转染人白血病细胞系K562,检测转染细胞中JTV1基因mRNA和蛋白的表达水平及其对K562细胞增殖的影响。方法从人外周血单个核细胞中克隆JTV1基因,并将其插入pcDNA3.1表达载体中,构建真核重组表达质粒pcDNA3.1-JTV1,经脂质体介导转染K562细胞,采用RT-PCR和Western blot法鉴定转染细胞中JTV1基因mRNA和蛋白的表达水平;MTT法检测JTV1稳定表达对K562细胞增殖的影响。结果重组表达质粒pcDNA3.1-JTV1经双酶切及测序证实,目的基因已插入质粒中;人JTV1基因能在K562细胞中稳定表达;JTV1具有抑制K562细胞增殖的作用。结论已成功构建了JTV1基因真核表达质粒,并获得了稳定表达人JTV1基因的K562细胞克隆,为进一步研究人JTV1基因的功能及其与白血病细胞增殖及凋亡的相关性提供了细胞模型。     

幽门螺杆菌粘附素hpa A和霍乱毒素B亚单位ctx B融合基因的原核表达

目的　构建幽门螺杆菌粘附素 (hpaA)和霍乱毒素B亚单位 (ctxB)融合基因的原核表达载体 ,诱导表达并进行纯化。方法　用PCR扩增hpaA和ctxB两个目的基因片段 ,克隆至同一pQE 30表达载体中 ,构建含双基因的表达质粒pQE hct,转化E .coliDH5α ,经IPTG诱导表达融合蛋白HCT ;经Westernblot分析其免疫原性 ,采用镍离子柱进行纯化。结果　经测序HCT融合基因片段由 116 1bp组成 ,为编码 387个氨基酸残基的多肽。经SDS PAGE分析相对分子质量约为 4 0 0 0 0。可溶性蛋白占菌体总蛋白的 2 5 %以上 ,经亲和层析后可获得纯度为 92 %以上的重组融合蛋白。经Westernblot检测可被Hp全菌抗血清和CT抗血清识别。结论　已成功构建融合蛋白HCT的原核表达载体并能高效表达 ,为研制Hp口服疫苗提供依据     

原发性开角型青光眼致病基因MYOC真核表达质粒的构建及其在COS-7细胞中的表达

目的构建原发性开角型青光眼(POAG)致病基因MYOC的真核表达质粒,并在COS-7细胞中表达MYOC蛋白。方法用RT-PCR法扩增人眼组织(角膜缘)MYOC基因cDNA,纯化回收后,克隆入pGEM-T载体,再亚克隆入真核表达质粒pEGFP-N3,构建重组表达质粒pEGFP-N3-MYOC,转染COS-7细胞,用荧光显微镜观察MYOC蛋白在COS-7细胞中的表达,Western blot分析MYOC蛋白分泌特点。结果经酶切和DNA测序鉴定,证实重组表达质粒pEGFP-N3-MYOC构建正确,荧光显微镜观察MYOC蛋白能在COS-7细胞中表达,并且定位在细胞质中,而绿色荧光蛋白分布在整个细胞内。Western blot结果显示,MYOC蛋白能分泌到细胞外。结论已成功构建MYOC基因真核表达质粒,并能在COS-7细胞中表达MYOC蛋白,为进一步研究POAG发病机制奠定了基础。