强信号肽的使用是提高蛋白分泌效率的关键,筛选强信号肽具有重要的应用价值.本试验以大肠杆菌-谷氨酸棒杆菌基础穿梭载体pAU2为骨架,以枯草芽孢杆菌的碱性丝氨酸蛋白酶AprE为报告蛋白,分别使用强启动子tac-M和谷氨酸棒杆菌核糖体结合位点一致序列来控制报告基因的转录和翻译,成功构建谷氨酸棒杆菌信号肽探测载体pAU20.分别对来自枯草芽孢杆菌、地衣芽孢杆菌、以及谷氨酸棒杆菌中共16种Sec型信号肽编码序列进行PCR扩增,与pAU20连接,转化谷氨酸棒状杆菌,获得重组信号肽探测载体工程菌株.工程菌株摇瓶发酵培养物上清液AprE酶活测定结果表明,C.glutamicum/pAU20-Vpr、C.glutamicum/pAU20-BprA上清液比酶活分别为180.02和167.6 U/mg蛋白质,均显著高于C.glutamicum/pAU20-cgr2070上清液比酶活(105.09 U/mg蛋白质);C.glutamicum/pAU20-oppA、C.glutamicum/pAU20-Epr、C.glutamicum/pAU20-cgr2063和C.glutamicum/pAU20-ywaD上清液比酶活分别为96.68、86.18、82.52和80.56 U/mg蛋白质,均稍低于C.glutamicum/pAU20-cgr2070上清液比酶活.这些结果表明,信号肽Vpr、BprA、cgr2070、oppA、Epr、cgr2063和ywaD都属于谷氨酸棒杆菌强信号肽.

鉴定强启动子可为谷氨酸棒杆菌(Corynebacterium glutamicum)代谢工程育种及重组蛋白生产提供高效的基因表达调控元件,但谷氨酸棒杆菌内源性强启动子的鉴定鲜有报道.本试验进行谷氨酸棒杆菌ATCC13032菌株细胞全蛋白二维电泳并对高表达蛋白质斑点进行质谱分析,选取分子量大小约为30 kDa、等电点约为4.6的斑点作为研究对象,分析其所对应蛋白编码基因的启动子.启动子预测结果表明,谷氨酸棒杆菌Cgl0864基因启动子P0864转录活性强.将强启动子Ptac-M和启动子P0864分别插入启动子探测载体pDXW-11,转化ATCC13032菌株感受态细胞,获得工程菌株C.glutamicum/pDXW-11-Ptac-M和C.glutamicum/pDXW-11-P0864.氯霉素耐受性试验结果显示,菌株C.glutamicum/pDXW-11-Ptac-M和C.glutamicum/pDXW-11-P0864的氯霉素耐受性分别为30和40μg/mL;报告蛋白CAT氯霉素酰基转移酶活性检测结果显示,C.glutamicum/pDXW-11-Ptac-M和C.glutamicum/pDXW-11-P0864菌株细胞抽提物上清液CAT蛋白氯霉素酰基转移酶比活力分别为4.50和6.12 U/mg;氯霉素乙酰基转移酶基因cat转录水平的荧光定量PCR试验检测结果显示,cat基因在启动子P0864的控制下,其转录水平是在Ptac-M控制下转录水平的1.84倍.以上结果表明,谷氨酸棒杆菌Cgl0864基因启动子P0864是强启动子.

干扰素γ诱导蛋白30基因(IFI30)能够在特异抗原递呈细胞中组成性表达,在其他细胞中能被IFN-γ诱导表达,IFI30在MHC-Ⅱ限制性抗原加工递呈过程中起关键性作用.为了构建猪IFI30表达载体并验证其在体外细胞表达情况.本试验从猪肺脏组织中扩增出IFI30中长度741 bp的CDS区,将其克隆到带有红色荧光的真核表达pEF1a-IRES-DsRed-Express2上,用脂质体转染法转染到Marc145细胞中,用RT-PCR以及Western blot检测其mRNA表达量和蛋白表达量.结果表明,成功构建出猪IFI30表达载体并在Marc145细胞中高效表达.

miRNAs are involved in various biological processes, such as host-virus interactions and antiviral immunity. In this study, we investigated the role of miR-29 on porcine reproductive and respiratory syndrome virus (PRRSV) replication and its target genes. At first, miR-29a/b-1/c expression was detected when porcine alveolar macrophages (PAMs) were infected with PRRSV at different infective doses by real time-quantitative polymerase chain reaction (RT-qPCR). The result showed that miR-29a/b-1 expression significantly increased after 6 h (p < 0.01), with the peak around 24 h, miR-29c expression in each period of PRRSV infection was very low. Then, pre-miR-29a/b-1 lentiviral vectors were constructed. Absolute RT-qPCR analysis showed that PAMs transfected with pre-miR-29a/b-1 lentiviral vectors significantly promoted PRRSV replication in PAM within 24 h (p < 0.01). The expression of the target genes (AKT3, TP53INP1, and RPS6KB1) of miR-29a significantly reduced (p < 0.01). Western blot analysis showed that AKT3 and TP53INP1 are reduced at miR-29a overexpression. To further validate the interaction between miR-29a and its target gene sites, the luciferase assay results demonstrated that miR-29a interacted with AKT3 3'UTR 1676 and 1261 sites, leading the inhibition of luciferase expression. Our findings support that miR-29a could promote PRRSV replication during early stage of virus infection in vitro and AKT3 could be the target gene of miR-29a.

To verified the target genes of miR-34c, bioinformatics software was used to predict the targets of miR-34c. Three possible target genes of miR-34c related to spermatogenesis and male reproductive development: zinc finger protein 148 (ZNF148), kruppel-like factor 4 (KLF4), and platelet-derived growth factor receptor alpha (PDGFRA) were predicted. Then, the expression of miR-34c and its target genes were detected in swine testicular tissue at different developmental stages by quantitative polymerase chain reaction. The results suggested that the expression of PDGFRA has the highest negative correlation with miR-34c. Then immunohistochemical staining was done to observe the morphology of swine testicular tissue at 2-days and 3, 4, 5-months of age, which indicated that PDGFRA was mainly expressed in the support cells near the basement membrane during the early development stages of testicular tissue, but that the expression of PDGFRA was gradually reduced in later stages. Therefore, western blot analyzed that the highest expression of PDGFRA was generated in 2-days old testicular tissues and the expression levels reduced at 3 and 4-months old, which correlated with the results of immunohistochemical staining. In conclusion, PDGFRA is a target gene of miR-34c.

microRNAs (miRNAs) represent a newly identified class of nonprotein-coding ∼ 22 nt small RNA that plays important roles in multiple biological processes by degrading targeted mRNA or repressing mRNA translation. This study observed the morphology of swine testicular tissue at different developmental stages (including 1-day old, 1-7 month old) by Hematoxylin-eosin staining. We also examined the expression of miR-499 and its target genes (CYLC1, DMRT1, QKI, XRN2, ZNF313) in samples of tissue slices using quantitative reverse-transcription polymerase chain reaction, which showed that miR-499 had a significant negative correlation with QKI gene. Then, the proteins of QKI gene expression were determined by western blot, which were consistent with results of quantitative polymerase chain reaction (qPCR) detection. Therefore, the luciferase reporter gene system was used to verify correlation between miR-499 and QKI gene. Activity of luciferase was significantly lower in miR-499 co-transfected with pmiR-RB-REPORT-QKI-WT group than the miR-499 co-transfected with pmiR-RB-REPORT-QKI-mut/si groups, indicating that target sequence of miR-499 existed in 3'UTR of QKI gene. Furthermore, the expressions of miR-499 and QKI were detected in testicular cells that were transfected with miR-499, miR-499 negative control and untreated. The results showed that the diameter of convoluted seminiferous tubule growth increased with age. Significantly different expressions of miR-499 and its target genes were found in swine testicular tissue at different developmental stages (p<0.05), overexpressing miR-499 analysis, suggesting that miR-499 was negatively correlated to the expression of QKI (p<0.05). In conclusion, QKI is a target gene of miR-499.

为了研究TLR7在病毒入侵时是否起到重要的抗原递呈作用,试验采用实时荧光定量PCR技术检测禽流感感染鸡和SPF鸡的免疫器官及发病器官中TLR7的表达水平差异情况.结果表明:TLR7基因在禽流感感染组和SPF组肺脏、气管上、气管下、脾脏、法氏囊、胸腺中均有表达,在不同组织中表达差异极显著,在感染组中的表达量明显高于SPF组.说明TLR7在禽流感病毒感染鸡的过程中表达量发生了改变.

生物信息学方法预测出miRNA-375可能的靶基因为DIAPH2和QKI,实时荧光定量法检测了靶基因在1日龄、1～7月龄军牧1号白猪睾丸组织中的表达量.结果表明,3月龄前猪睾丸组织中miRNA-375表达量极低,4～7月龄表达水平逐渐上调,差异显著.预测靶基因DIAPH2在1～7月龄呈显著下调趋势,OKI基因在1～7月龄睾丸组织中表达水平呈显著上调趋势.经SPSS13.0分析可知,不同时期睾丸组织中miRNA-375与2个预测靶基因DIAPH2和QKI的相关系数r分别是-0.396(P＜0.05),0.411(P＜0.05),2个预测靶基因DIAPH2和QKI之间的相关系数是0.151(P＞0.05),靶基因DIAPH2和QKI无明显联系,初步证明DIAPH2可能是miR-NA-375的靶基因,为探索猪睾丸组织发育提供了一定的研究基础.

Interferon, gamma-inducible protein 30(IFI30)plays a key role in MHC classⅡ-restricted antigen processing and MHCⅠ-restricted cross-presentation. In our study, PAMs were infected by PRRSV of three different concentrations, and the levels of IFI30 mRNA expression of different time were detected by qPCR. The results showed that the IFI30 expression differences were not significant when PAMs were infected by three different concentrations,0.08 MOI,0.10 MOI and 0.12 MOI(P 0.05), respectively. The expression of IFI30 infected by PRRSVafter1.5 hwas reduced,butthe difference was not significant(P 0.05), and then the expression of IFI30 was increased, the expression difference was very significantwhen PAMs were infected by PRRSV after 12 h, 24 h and 36 h(P 0.01), but then the expression of IFI30 was reduced(P 0.05). The results indicated that the IFI30 expression change in the process of PRRSV infecting PAM may be one of the mechanisms which interact between PRRSV and host.

To compare the detection of the blood physiological and biochemical indexes in lactation and mature stages for pigs from three different genetic backgrounds,104 healthy pigs in lactation(at one-month old) and mature stages(at seven-month old) were selected under the same environment,including 41 Chinese experimental miniature pigs,38 Junmu No.1 white pigs,and 25 Duroc,and then anterior vena cava or ear vein blood samples were collected to analyze 28 blood indexes.A general linear model analysis of variance was used to analyze the measured data using SAS9.2 statistical software.The results showed that most of the blood physiological and biochemical indexes were affected by developmental stages,breeds,and the interactions of them.The developmental stages had the greatest impact on blood physiological and biochemical indexes,followed by the interactions of breeds and developmental stages,breeds and gender.The differences between breeds on the levels of red blood cell(RBC) count,hemoglobin(Hb)content,and hematocrit(HCT) might be related to the behavioral characteristics of pigs,and creatinine(CREA),triglyceride(TG),and total serum cholesterol(CHOL) were related to the content of pig lean meat and fat percentage.

DNA methylation is one of the main epigenetic modification mechanisms in eukaryotic organisms,playing a crucial role in the regulation of gene expression.In this study,we put F1 hybrid with Laiwu pigs and Yorkshine as research object,used the fluorescence-labeled methylation-sensitive amplified polymorphism(F-MSAP) method and used 16 pairs of selective primers to assess the extent and pattern of cytosine methylation in the muscle.The incidence of DNA methylation was approximately 57.20% in the muscle of direct cross and total of 2 031 sites were found to be methylated at cytosines.53.50% in the reciprocal cross and total of 1 905 sites were found to be methylated at cytosines.As type Ⅱ of methylation status,the reciprocal cross was higher than the direct cross significantly(P0.05).As type Ⅲ of methylation status,the direct cross was higher than the reciprocal cross exthreme significantly(P0.01).

MicroRNAs(miRNAs) are short(-22 nucleotides) non-coding RNA molecules that play important roles by repressing translation or cleaving RNA transcripts.Some reports showed miRNA-34b is an important regulator in germ cell differentiation process of male humans and mice.We detected the different expressions of miR-34b of testicular tissue by real time quantitative PCR(RT-PCR) in Junmu No.1 white pig among the whole developmental stages.The results showed the expressions of miRNA-34b keep very low level before 3-month-old,rising dramatically from 5-month-old,reaching a peak at 7-month-old,and then descend apparently.Overview the expression trend of miRNA-34b in the whole sexual development processes,it runs a rule that first rising and then descending.The conclusion is anticipated that miR-34b keep a high possibility correlation with sexual development of male pig.

FUT1 and RYR1 are main effect genes for disease resistance in swine 6 chromosome q11-12. In order to in- vestigate the association of them in Junmu-1 white pigs, 177 pigs were randomly selected to detect genotypes of FUT1A and RYR1T by PCR-RFLP. Results showed allele frequencies and gene frequencies,linkage disequilibrium, and correlation of them. FUT1A frequency was 0. 395 ,RYR1T was 0. 765. The linkage disequilibrium parameters LD = 0. 010 7, the correlation coefficient r= 0. 093 6, both close to zero. The conclusion indicates no genetic association between FUT1 and RYR1 ,and supports the combined selection of the two resistance genes in Junmu-1 population.

540 healthy Luhua chickens(1-day old)with similar body weight were randomly separated into 3 groups,3 replicates per group,60 birds per replicate.Group Ⅰ,GroupⅡ,GroupⅢ were fed high dietary nutrition,medium dietary nutrition and low dietary nutrition separately.The results showed that:(1) Growth performance: the medium dietary nutrition can meet the need of Luhua chicken's growth.The high dietary nutrition had obviously growth promoting effect for only 1～28 d.The low dietary can not meet the growth need of Luhua chicken.(2) Slaughter performance: the live weight,slaughter rate,half-eviscerated rate,and carcass net rate were significantly greater than the low dietary group,slightly higher than the high dietary group.The chest and leg muscle percentage of high dietary group was higher than the other two groups.The abdominal fat rate and thick leather fat of medium group was the highest,but there were no significant differences.In short,recommend fed Luhua with the medium dietary nutritions.

试验选用900只1日龄健康芦花鸡,随机分成3组:对照组(基础日粮)、试验Ⅰ组(基础日粮+0.1 g/kg抗菌肽)和试验Ⅱ组(基础日粮+0.1 g/kg抗菌肽+0.5亿个/kg酵母活菌).结果表明:与对照组相比,日粮中添加抗菌肽和抗菌肽制剂可极显著提高芦花鸡末质量和平均日增质量,极显著降低平均日耗料和料肉比；对42、56和70日龄芦花鸡血清尿素氮、白蛋白和球蛋白质量浓度均无显著影响；抗菌肽制剂和抗菌肽能分别显著提高42和70日龄血清总蛋白质量浓度；抗菌肽对42、56和70日龄血清三酰甘油和总胆固醇水平均无显著影响；抗菌肽制剂显著提高42日龄血清总胆固醇水平.

HABP4 participates in a variety of important physiological and biochemical processes in mammalian, but only few studies have involved its exact physiological function and mechanism of action. In this study, using real-time quantitative PCR technology detected HABP4 gene expression levels in the muscle tissue of hybrid progeny of Large White and Laiwu pigs. The results showed that HABP4 gene was expressed in all longissimus muscle tissue detec- ted. And the expression level is different of longissimus muscle tissue in the two hybrid generation. HABP4 gene ex- pression in the longissimus muscle of the quadrature generation was significantly higher than anti-cross generation. The test provides the evidence of the molecular genetics basis to improve swine carcass traits, and laid the foundation of later in-depth study on the biological role and mechanism of the HABP4 gene in different pig breeds.

Luhua,Huang,Ma chickens were used as experimental population. RT-PCR technique was used to study gene expression in heart fatty acid-binding protein （H-FABP）gene at 110 days. The results showed that the expres- sion of H-FABP gene in Ma chicken was significantly lower than that of Huang and Luhua chickens by 174.8%, 170.070% （P〈0.05）, there was no significant differences between Luhua and H uang chickens, which presented a significant difference of varieties.

<正>优质肉鸡由地方优良品种鸡选育而成,保留了农村土鸡的特点,具有低脂肪、高蛋白、低胆固醇、口感好等优点。近年来,随着人们生活水平的提高,消费市场逐渐扩大。笔者选取了芦花鸡、黄鸡和麻鸡三个优质肉鸡品种,对其在北方环境条件下饲养时的肌肉品质进行了比较研究,旨在优化、筛选出适合北方发展且肌肉品质较好的优质肉鸡品种。

This study was designed to investigate the single nucleotide polymorphism(SNP) by PCR-SSCP of PRKAG3 gene in 87 plymouth rock,at the same time the correlation between the polymorphism and meat quality traits.The result of PCR-SSCP showed that there were three genotypes(TT,TG and GG).The correlation analysis revealed that polymorphism had a extremely significant effect on PH,the plymouth rocks with GG genotype had lower PH than the GT and TT genotype(P<0.01),at the same time,the GT genotype lower than the TT genotype(P<0.05).other meat quality traits of plymouth rock were not significant influence on different genotypes(P>0.05).It could be preliminarily concluded that PRKAG3 gene was probably a major gene or a QTL linked gene which associated with PH and can be chosen as the molecular markers of meat quality in plymouth rock.