载脂蛋白CⅡ基因在猪肝脏组织中的发育性表达研究

试验旨在探究载脂蛋白CⅡ(apolipoprotein Ⅱ,ApoCⅡ)基因mRNA在马身猪和大白猪肝脏组织中的发育性表达规律,揭示ApoCⅡ基因表达水平与猪脂质代谢之间的关系。采用实时荧光定量PCR技术检测了马身猪和大白猪1~180日龄(1、30、60、90、120、150和180日龄)肝脏组织中ApoCⅡ基因mRNA表达。结果表明,马身猪和大白猪肝脏组织ApoCⅡ基因mRNA发育性变化趋势存在差异,马身猪ApoCⅡ表达量从1日龄到60日龄逐渐降低,在90日龄时上升,之后逐渐降低;而大白猪从1日龄到150日龄呈现逐渐降低趋势,180日龄时回升。1日龄时,马身猪和大白猪肝脏中ApoCⅡ基因mRNA的表达量差异不显著(P>0.05),在其他阶段,两品种间的表达量存在显著或极显著差异(P<0.05或 P<0.01)。这表明肝脏中ApoCⅡ mRNA的表达具有显著的日龄依赖性和明显的品种差异性,其表达可能对猪脂质代谢有显著影响。     

Tissue Expression Profile and Differential Expression of LTβR Gene in Sutai Piglets of Different Ages

Dynamic changes of LTβR expression levels in 11 tissues (heart, liver, spleen, lung, kidney, stomach, muscle, thymus, lymph node, duodenum and jejunum) of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 were compared and analyzed by the Real-time PCR method, which aimed to provide theoretical basis for further investigate the relationship between LTβR gene and pathogenicity of E.coli F18.The results revealed that the LTβR expression levels were higher in the liver, spleen, lung, kidney, stomach, lymph node, duodenum and jejunum, and showed obvious age-dependent expression differentiation.The LTβR expression levels in the lymph node, duodenum, and jejunum were extremely significant higher in 8 days old piglets than in the other age stages (P<0.01), and the expression levels were extremely significantly higher in the lungs of 8 days old piglets than in 35 days old piglets (P<0.01) and significantly higher than 30 days old piglets (P<0.05).In the liver tissue, the expression level was extremely significant higher in 35 days old piglets than in other age stages (P<0.01).In the stomach tissue, the expression level was significantly higher in 35 days old piglets than in 18 days old piglets (P<0.05).The results speculated that intestinal immune barrier of piglets formed rapidly around 8 days old and the higher LTβR expression could contribute to the resistance to E.coli F18.     

Association Analysis between Differences of GnRHR-Ⅱ Gene Expression with Reproduction Traits Heterosis in Duck Populations

The objective of this study was to clone the sequence of duck type Ⅱ gonadotropin releasing hormone receptor (GnRHR-Ⅱ) gene, and to analyze the association of its expression levels with reproduction trait heterosis. RT-PCR method was used to clone GnRHR-Ⅱ gene fragments, and the expression levels during early laying days and laying fastigium period in Gaoyou duck, Jinding duck and crossed populations were tested by Real-time PCR method. Sequencing and homology analysis showed that the cloned duck GnRHR cDNA was about 500 bp in length, and it belonged to the avian type Ⅱ GnRHR isoforms. It was 96% identical to the cGnRHR-Ⅱ sequence reported in domestic chicken. From the early laying days to laying fastigium period, expression levels of GnRHR-Ⅱ gene in pituitary increased for Jinding duck, Gaoyou duck and crossed populations, with the highest expression levels in Jinding×Gaoyou hybrid group (P<0.01) . The expression levels of GnRHR-Ⅱ gene in hypothalamus also increased for Jinding duck, Gaoyou duck, however, it decreased in two crossed populations. The expression levels of GnRHR-Ⅱ gene in ovary were significantly higher for Jinding duck and two crossed populations than Gaoyou duck (P<0.01), but it decreased during laying fastigium period. Compared to Jinding duck and Gaoyou duck breeds, the two crossed populations had positive heterosis in 42-week production and 42-week egg fertilization percent, and negative heterosis in the first laying age and 42-week fertilized egg hatching rate traits. It suggested that the short-lived higher expression of ovary GnRHR-Ⅱ gene before early laying days be correlated with heterosis of the first laying age and 42-week production traits, and its higher expression levels in pituitary could aid to keep laying fastigium.     

Construction and High Level Expression of Prokaryotic Expression Vector of Pig Interferon-δ Gene

Interferon (IFN) had recieved much more attention because of its broad-spectrum antiviral, antitumor activity and immune regulation.One pair of primers were designed for amplifying pig IFN-δ gene with PCR method according to the relevant sequence from GenBank.The IFN-δ gene was cloned into prokaryotic expression vector, and the recombinant plasmid was obtained.We transformed the recombinant plasmid into E.coli Transetta BL21(DE3) strain and the protein expression was identified by SDS-PAGE and Western blotting.The results showed that pET-30a-DsbA-IFN-δ recombinant protein was 20 ku and expressed mainly in the form of inclusion body, these expressed protein could specific react with His-tag monoclonal antibody.     

CREB-H基因在猪不同组织中表达谱及肝脏中的表达规律

本研究旨在分析环腺苷酸应答元件结合蛋白H（cyclic AMP-responsive element-binding protein 3-like 3,CREB-H）在猪不同组织中的表达谱及其在马身猪和大白猪肝脏中的发育性表达规律。采用实时荧光定量PCR和Western blotting技术检测1日龄猪12个组织（心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、小脑、下丘脑、背最长肌、股肌和腰肌）中CREB-H基因的表达谱,以及CREB-H在1、30、60、90、120、150和180日龄马身猪和大白猪肝脏中的表达规律。结果显示,CREB-H基因mRNA在马身猪的12个组织中广泛表达,其中在肝脏和小肠中高表达;CREB-H蛋白在肝脏组织中的表达量显著高于其他组织（P<0.05）,在心脏、脾脏和小脑中不表达。猪肝脏CREB-H基因mRNA和蛋白的发育表达受日龄、品种、品种与日龄相互作用的影响（P<0.01）。马身猪和大白猪肝脏中CREB-H基因mRNA和蛋白的表达量均在1日龄时达到最大值。在各发育阶段,马身猪CREB-H蛋白的表达量均极显著高于大白猪（P<0.01）,且CREB-H主要在猪肝脏中表达。CREB-H在两猪种肝脏中的表达存在时空差异,可能与猪在不同发育期的脂质代谢能力有关,本试验结果为研究猪的脂质代谢调控机制提供一定的理论依据。     

Cloning of Inhibin-α Gene and its mRNA Expression Pattern in the Ovary of Congjiang Xiang Pig

To reveal the relationship between inhibin-α(INHA) gene and the reproductive traits of Congjiang Xiang pig, INHA gene was cloned and sequenced taking the genomic DNA of Congjiang Xiang pigs as templates by polymerase chain reaction(PCR) method.The polymorphisms of INHA gene were tested in Congjiang Xiang pig populations with high-litter size and low-litter size using allele-specific PCR(AS-PCR) method.The expression profile of INHA gene in ovaries was detected from Congjiang Xiang pigs with high-litter yiled or low-litter yiled by Real-time PCR method.The complete coding region of INHA gene was 1095 bp in length, which coded for 364 amino acid residues.Compared with the known sequence, two candidate sites, G359A and A373G, were found out from exon 2 region of INHA gene in Congjiang Xiang pig.After investigation for the two sites in a large population, the frequency of alleles between two populations was not significant and without obvious relativity with the litter yiled of Congjiang Xiang pig.However, the INHA mRNA level in the ovary of Congjiang Xiang pig with high-litter yiled was higher than that with low-litter yiled.It suggested that INHA gene was much conserved, INHA gene expression level might be concerned for the regulation of ovary growth and follicle development in Congjiang Xiang pig breed.     

MYNN基因在猪不同组织及肌肉中的表达规律

试验旨在研究myoneurin（MYNN）基因在猪不同组织中的表达特征及其在肌肉（背最长肌、股二头肌和腰大肌）、小脑、肝脏、胰脏、肾脏、胃、脾脏、肺脏组织中的发育性表达规律。采用实时荧光定量PCR技术研究猪MYNN mRNA在90日龄大白猪和马身猪的心脏、肝脏、脾脏、肺脏、肾脏、小脑、小肠、胰脏、胃、股二头肌及脂肪共11个组织中的表达谱,以及在大白猪和马身猪1、90、180日龄3个发育阶段的肌肉、小脑、肝脏、胰脏、肾脏、胃、脾脏、肺脏组织中的发育性表达规律。结果表明,MYNN在猪的各种组织中广泛表达,且各组织间表达差异显著或极显著（P < 0.05;P < 0.01）;MYNN在大白猪和马身猪的肌肉、小脑、肝脏、胰脏、肾脏、胃、脾脏、肺脏组织中的不同发育阶段表达差异显著或极显著（P < 0.05;P < 0.01）,并具有特定规律,由此推测其可能在猪的这几种组织中发挥重要作用。MYNN基因的表达与组织、日龄及品种的遗传背景有关。本试验为研究猪MYNN基因的生物学功能提供了依据,但还需要深入的研究来探索其作用的具体机制,尤其是在骨骼肌发育中的调节机制。     

脂肪酸合成酶基因mRNA在马身猪和大白猪3种组织中的发育性表达研究

为探究脂肪酸合成酶(fatty acid synthase,FAS)基因在猪不同组织中的发育性表达规律,本研究采用实时荧光定量PCR技术检测马身猪和大白猪7个阶段(初生、30、60、90、120、150和180日龄)肝脏、背最长肌和背部皮下脂肪3种组织中FAS基因mRNA的相对表达量。结果表明,品种间比较,FAS基因mRNA在马身猪和大白猪肝脏、背最长肌和背部皮下脂肪组织各生长发育阶段中的表达差异均达到显著或极显著(除肝脏组织初生阶段和背部皮下脂肪组织120日龄阶段)(P<0.05;P<0.01)。FAS基因mRNA在大白猪组织间的表达差异与生长发育相关,150和180日龄阶段,背部皮下脂肪组织中表达量极显著高于肝脏和背最长肌组织(P<0.01),初生、30日龄和90日龄阶段,背最长肌中的表达量极显著高于肝脏和背部皮下脂肪组织(初生阶段无脂肪组织样)(P<0.01);而马身猪整个发育过程中,背最长肌组织表现为优势组织,极显著高于其他2种组织(除120日龄阶段外)(P<0.01),脂肪组织表达量次之,肝脏组织中表达量较少。品种、日龄、组织及品种与日龄、组织与日龄的互作效应对FAS基因mRNA的相对表达量均有极显著影响(P<0.01)。FAS基因直接参与脂肪酸的合成,对猪肉质性状的遗传改良具有重要意义。     

The Expression Characteristics of MYNN Gene in Different Tissues and Muscles of Pig

This study was aimed to investigate the expression characteristics of myoneurin (MYNN) gene in different tissues and its developmental expression in muscles (longissimus dorsi, biceps femoris and psoas major), cerebellum, liver, pancreas, kidney, stomach, spleen and lung tissues of pigs. The expression characteristics of MYNN mRNA in 11 different tissues including heart, liver, spleen, lung, kidney, cerebellum, small intestine, pancreas, stomach, biceps femoris and fat of Large White pigs and Mashen pigs at the age of 90 days and the developmental expression patterns in muscle, cerebellum, liver, pancreas, kidney, stomach, spleen at three strages (1, 90, 180 days) of Large White pigs and Mashen pigs were studied by Real-time PCR. The results showed that MYNN was widely expressed in various tissues of pigs, and there was significant difference among the tissues(P < 0.05; P < 0.01); The expression of MYNN in muscle, liver, pancreas, cerebellum, kidney, stomach, spleen, lung tissues were significant difference at three development stages of Large White pigs and Mashen pigs(P < 0.05; P < 0.01), and also had a specific rule, which indicated that it may play an important role in these pig tissues. The expression of MYNN gene could related to the tissue, age and the genetic background of breeds. The results of this study provided a better understanding of the biological functions of pig MYNN. Further studies are required to determine its molecular mechanisms, especially in the regulation of skeletal muscle development.     

Effects of Compound Chinese Herbal Medicinal Polysaccharides on Immune Signal Molecules Expression in Different MHC B-LβⅡ Genotypes Chickens Lymphocyte

This study was aimed to explore the effect of compound Chinese herbal medicinal polysaccharides (cCHMPS) on immunomodulatory in different MHC B-LβⅡ genotype chickens.MHC B-LβⅡ genotype was analyzed by PCR-SSCP method in 500 chickens.Collected the blood from different MHC B-LβⅡ genotype chickens,isolated peripheral blood lymphocytes,and added cCHMPS with the final concentration of 200,100,75,50,25 and 0 μg/mL cocultured for 24 h.Then the lymphocyte supernatant cAMP,cGMP,Ca²⁺,NO and iNOS content were detected by ELISA.The results showed that cCHMPS could increase cAMP,cGMP,Ca²⁺,NO and iNOS levels in chicken lymphocyte supernatant compared with control group in each MHC B-LβⅡ genotype chickens.And when the concentration of cCHMPS was 50 μg/mL,cAMP,Ca²⁺,NO and iNOS levels of AA and BC genotypes chicken lymphocyte supernatant were higher than that of the same genotype chicken,cGMP level of BC genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 75 μg/mL,cGMP level of AA genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 100 μg/mL,cAMP,cGMP,Ca²⁺,NO and iNOS levels of BB genotype chicken lymphocyte of the supernatant were higher than that of the same genotype chicken.cCHMPS could increase cAMP,cGMP,Ca²⁺,NO and iNOS levels in different MHC B-LβⅡ genotypes chickens,and the cCHMPS optimum immunomodulatory doses were different in each MHC B-LβⅡ genotypes chickens.     

Pax7基因在猪不同组织中的表达特征及其在背最长肌中的发育性表达规律研究

本试验旨在探究Pax7(paired box 7)基因在猪不同组织中的表达特征及其在背最长肌中的发育性表达规律.采用实时荧光定量PCR(qRT-PCR)技术和Western blotting技术检测了Pax7基因在1日龄马身猪心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、下丘脑、小脑、背最长肌、腰大肌、股二头肌12种组织中的mRNA和蛋白质表达谱,以及马身猪和大白猪从出生1日龄到180日龄7个发育阶段(1、30、60、90、120、150、180日龄)背最长肌中的发育性表达规律.结果表明,Pax7基因mRNA在背最长肌、腰大肌、股二头肌、下丘脑和小脑组织中表达,而Pax7蛋白仅在背最长肌、腰大肌和股二头肌中表达.背最长肌中Pax7基因mRNA和蛋白质的发育性表达趋势在马身猪和大白猪中基本一致.在mRNA水平上,30日龄的表达量最高,极显著高于其他日龄(P <0.01);1日龄的表达量次之;其他日龄维持低表达的稳定状态.Pax7蛋白表达量在1日龄时最高,极显著高于其他日龄(P <0.01);30日龄次之;其他日龄维持低表达的稳定状态.从出生1日龄到180日龄,马身猪背最长肌中Pax7蛋白的表达量均显著或极显著地高于大白猪(P <0.05;P <0.01).Pax7 基因的表达与组织、日龄及品种的遗传背景有关.     

Study on the Expression Profile of Pax7 Gene and its Developmental Expression in Longissimus Dorsi of Pigs

The aim of this study was to investigate the expression profile of Pax7 (paired box 7) gene in different tissues and its developmental expression in longissimus dorsi of pigs.The expression profiles of Pax7 gene at the levels of mRNA and protein in 12 different tissues including heart,liver,spleen,lung,kidney,stomach,small intestine,hypothalamus,cerebellum,longissimus dorsi,biceeps femoris,psoas major of Mashen pigs at birth and the developmental expression patterns in longissimus dorsi at seven stages (1,30,60,90,120,150,and 180-day old) of Mashen and Large White pigs were studied by quantitative Real-time PCR and Western blotting.The results showed that the Pax7 mRNA was expressed in longissimus dorsi,biceeps femoris,psoas major,hypothalamus,and cerebellum,wherease the Pax7 protein was only expressed in skeletal muscles of longissimus dorsi,biceeps femoris,and psoas major.The developmental expression patterns of Pax7 mRNA and protein in Mashen pig was basically in accordance with those in Large White pig.The mRNA expression amount of Pax7 gene in longissimus dorsi was the greatest at 30-day old (P <0.01),followed by the expression at 1-day old,and kept lower stable levels at other stages both in Mashen and Large White pigs.At the stages of 30-day,90-day,and 180-day old,Pax7 mRNA expression amounts in Mashen pig were significantly higher than those in Large White pig (P <0.01).The Pax7 protein expression at 1-day old was the greatest (P <0.01),followed by it at 30-day old,which were significantly greater than those at other stages,at which Pax7 expression was in lower stable levels.The Pax7 protein expression from one-day old to 180-day old in Mashen pig was significantly greater than those in Large White pig (P <0.05;P <0.01).The expression of Pax7 gene could associated with tissue,age,and background of pig breed.     

大白猪和山西黑猪三种肌肉JAZF1基因的发育性表达研究

本文旨在探究JAZF1基因mRNA在大白猪和山西黑猪肌肉组织中的发育性表达规律,为研究猪肌内脂肪的形成机理提供有力的理论参考。采用实时荧光定量PCR技术检测大白猪和山西黑猪在25d、70d和150d的背最长肌、腰大肌和股二头肌三种肌肉组织中JAZF1基因mRNA表达量。结果表明:随着日龄的增长,JAZF1基因在三种肌肉中呈现逐渐下降的表达趋势。大白猪25d腰大肌和股二头肌、70d股二头肌中的表达量显著高于山西黑猪(P<0.05)。三种肌肉中的表达量比较发现,JAZF1基因在两个品种腰大肌中表达量最高,其次是股二头肌,背最长肌表达量最低。结果表明JAZF1基因在肌肉发育和肌内脂肪沉积过程中起着很重要的作用。本研究结果可为探索肌肉发育及肌内脂肪沉积相关研究提供理论参考和研究基础。     

猪Fosl2基因克隆、结构预测及其mRNA发育性表达变化

本试验旨在克隆猪Fosl2基因CDS序列并对其进行分析,研究该基因在猪的不同阶段各种组织(心、肝、脾、肺、肾、背最长肌、背部脂肪)中的发育性表达规律。以豫南黑猪为材料,利用RT-PCR技术克隆猪Fosl2基因CDS区域,利用生物信息学方法预测蛋白结构,应用qRT-PCR检测豫南黑猪7~180日龄上述组织中Fosl2基因的发育性表达。结果表明,猪Fosl2基因的CDS区为984bp,编码327个氨基酸,跨膜序列为17~33位氨基酸,该肽链没有信号肽,为跨膜非分泌型蛋白,在122~187位氨基酸的位置存在亮氨酸拉链结构域,核苷酸和氨基酸序列同其他种属间的保守性很高。Fosl2基因拥有广泛的组织表达谱,在心、肝、脾、肾、肺、背最长肌和背部脂肪的不同发育阶段均有不同程度的表达,而其在肺中的表达量均高于同时期其他组织内的表达量,推测猪Fosl2基因可能与肺部组织的发育有关;其在背部脂肪不同发育阶段的变化趋势表明其可能参与调控猪脂肪的生成进而影响瘦肉率。本研究结果将为进一步研究猪Fosl2结构及其多功能奠定基础。     

过氧化物酶体增殖物激活受体α在猪肝脏组织中发育性表达研究

为了探究过氧化物酶体增殖物激活受体α(peroxisome proliferator activated receptor alpha,PPARα)基因在不同脂肪型猪肝脏组织中的发育表达规律,本研究采用实时荧光定量PCR方法,检测了马身猪和大白猪0、1、2、3、4、5和6月龄个体肝脏内PPARα基因mRNA的表达量。结果显示,马身猪和大白猪肝脏组织PPARα基因mRNA发育性变化趋势存在差异,大白猪PPARα 基因表达量呈现出前、后期高中期低的变化趋势,0月龄时表达量最高,3月龄时表达量最低,两者差异极显著(P<0.01),而马身猪表达量呈现前期高后期低的变化趋势,0月龄和1月龄的表达量极显著高于其他月龄(P<0.01)。上述结果揭示猪PPARα可能是肝脏脂质代谢的调控因子,为猪脂肪沉积的调控机制提供理论数据。     

Correlation Study of CARD15 Gene StyⅠPolymorphism and Susceptibility of Tuberculosis in Dairy Cows

The aim of this study was to investigate the association between CARD15 gene StyⅠpolymorphism and susceptibility to tuberculosis (TB) of dairy cows from Yunnan plateau.A total of 201 dairy cows (103 cases and 98 controls) from Kunming,Yuxi and Dali were used in this study.We designed a specific primer pair for PCR-RFLP analysis.By using PCR-RFLP genotyping and PCR sequencing,We analysized allele frequency and correlation between polymorphism of CARD15 gene and susceptibility to bovine tuberculosis in Yunnan province.The results showed that there were three genotypes in Sty Ⅰ enzyme site,AA,AG and GG.In cases,the genotype frequencies of AA,AG and GG were 0.03,0.42 and 0.55,respectively,while in controls were 0.01,0.05 and 0.94,and the allele frequencies of A and G were 0.24 and 0.76 in cases,while were 0.04 and 0.96 in controls.Then analysizing the genetic pattern of polymorphic loci,we found that the best genetic model was dominant and the frequency of AG-AA in cases (44.7%) was apparently higher than that in controls (6.1%) (OR(95% CI)=0.08 (OR< 1)),so extremely significant difference existed between cases and controls (P< 0.0001).The polymorphism of StyⅠenzyme cut site in CARD15 gene was significantly associated with the susceptibility to tuberculosis of Yunnan plateau dairy cows,and AG-AA might be the susceptible genotype to tuberculosis.     

Effects of Compound Chinese Herbal Medicinal Polysaccharide Extraction on NF-κB,TNF-α and IL-6 mRNA Expression in Different MHC B-Lβ Ⅱ Genotype of Chickens Lymphocyte

In order to explore the effect of compound Chinese herbal medicines polysaccharides(cCHMPS) on immunomodulatory in different MHC B-Lβ Ⅱ genotype chickens,200 White feather broiler were chosen and PCR-SSCP technique was applied to analyze the polymorphism of MHC B-Lβ Ⅱ gene. The peripheral blood were collected according to the different MHC B-Lβ Ⅱ genotype,and the lymphocytes were isolated from peripheral blood and the cCHMPS were added with a final concentration of 100,75,50 and 0 μg/mL for co-culturing 24 h.Then the expression of NF-κB,TNF-α,IL-6 mRNA in lymphocyte using Real-time PCR method were detected. The results showed that:Compared with the control group,different does of cCHMPS could significantly improve NF-κB,TNF-α,IL-6 mRNA expression levels in chicken with different MHC B-Lβ Ⅱ genotypes (P<0.05), and when the cCHMPS concentration was 50 μg/mL,the NF-κB,TNF-α,IL-6 mRNA expression levels in lymphocyte of AB and AA genotype chicken were significantly higher than that of other groups (P<0.05).The NF-κB,IL-6 mRNA expression levels of AC genotype chicken were significantly higher than the other groups (P<0.05). The TNF-α mRNA expression levels of AC genotype chicken were significantly higher than the other groups when cCHMPS was 100 μg/mL (P<0.05).There results indicated that the cCHMPS could stimulate NF-κB,TNF-α,IL-6 mRNA expression in different MHC B-Lβ Ⅱ genotype chickens,and the optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chicken.     

猪胸膜肺炎放线杆菌Ⅱ型分泌蛋白基因的克隆与表达

研究通过筛选猪胸膜肺炎放线杆菌3～8 kb随机基因组文库获得1株阳性克隆.测序拯救质粒得到序列,通过BLAST分析确定此片段为猪胸膜肺炎放线杆菌Ⅱ型分泌蛋白基因,预计成熟蛋白分子质量为45.716 ku.根据序列设计特异性引物PCR扩增该基因,分子克隆表达该Ⅱ型分泌蛋白.Western-blot结果表明,该蛋白能与猪胸膜肺炎放线杆菌康复期血清发生反应.