二花脸猪和大白猪睾丸IGF-Ⅰ和IGF-IR基因表达发育变化的研究

为了研究生长轴激素对猪繁殖性能发育的影响,随机选取0、3、20、30、90、120、180日龄纯种二花脸公猪和大白公猪各4头,屠宰并采取睾丸组织样,以18S rRNA为内标,用相对定量RT-PCR法研究睾丸IGF-Ⅰ和IGF-IR mRNA的表达及发育性变化。结果表明,二花脸猪与大白猪睾丸IGF-ⅠmRNA表达的发育模式在30日龄前完全相同,即随着日龄的增加而呈极显著增加（P〈0.01）;二花脸猪睾丸IGF-Ⅰ mRNA相对表达量在30-180日龄无显著变化;大白猪睾丸IGF-Ⅰ mRNA相对表达量在90日龄有所下降,而在120和180日龄又恢复到30日龄水平。二花脸猪睾丸IGF-Ⅰ mRNA相对表达量显著高于大白猪（P〈0.05）。二花脸猪与大白猪睾丸IGF-IR mRNA表达的发育模式不同。二花脸猪睾丸IGF-IR mRNA相对表达量在90-120日龄呈显著下降趋势（P〈0.05）;大白猪IGF-IR mRNA相对表达量在0日龄较高,随后显著下降（P〈0.05）,并在观察期内持续保持较低水平。二花脸猪IGF-IR mRNA相对表达量显著高于大白猪（P〈0.05）。睾丸IGF-Ⅰ mRNA和IGF-IR mRNA相对表达丰度呈极显著正相关（r=0.575,P〈0.01）。结论：（1）不同品种猪睾丸IGF-Ⅰ和IGF-IR mRNA表达具有特定的发育模式;（2）猪睾丸中IGF-Ⅰ和IGF-IR mRNA的协同表达可能对猪繁殖性能的发育有重要调节作用。     

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。     

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。     

猪背最长肌肌纤维类型的发育性变化及其品种和性别特点

4种肌球蛋白重链的异构体(MyHC Ⅰ、MyHC2a、MyHC2x、MyHC2b)分别特征性地对应4种不同类型的肌纤维(Ⅰ、2a、2x和2b)。选用生长速度快，但内质较差的瘦肉型猪大白猪和生长速度慢，但内质较好的肥胖型猪二花脸作为实验动物，采用相对定量RT-PCR方法，测定了背最长肌中Ⅰ、2a、2x和2b4种MyHC mRNA基因的表达，以探讨二花脸猪和大白猪背最长肌中Ⅰ、2a、2x和2b4种肌纤维类型比例的发育性变化，并分析了其品种和性别特点。结果表明：(1)3日龄MyHcⅠ型和2a型肌纤维的比例较高，2b型几乎没有分化；(2)从3日龄到20日龄，肌纤维组成发生了急剧的变化，MyHCⅠ型和2a型表达下调，比例下降，2b型肌纤维显著上升(P＜0．05)，且品种间无显著差异；(3)90日龄以后，二花脸公猪背最长肌中MyHCⅠ型和2a型的比例均显著高于大白猪，同时，大白猪背最长肌中酵解型肌纤维2b的比例增加迅速，显著地高于二花脸(P＜0.05)；(4)性别间比较，MyHCⅠ型纤维的比例无显著差异，90日龄和180日龄，二花脸母猪的MyHC2b的比例显著高于二花脸公猪(P＜0.05)。以上结果提示，大白猪和二花脸猪背最长肌肌纤维类型的差异主要表现在90日龄后，大白猪Ⅰ型纤维比例的显著下降和2b型纤维比例的显著增加与其肌肉的快速沉积有关；而二花脸猪背最长肌较高比例的MyHcⅠ型和2a型纤维与其优良的内质相关。     

五指山猪8种组织IGF2和IGFBP3基因表达的发育性变化研究

采用Q-PCR 技术检测了胰岛素样生长因子2(IGF2)和胰岛素样生长因子结合蛋白3(IGFBP3)基因在30、60、90、120 和150 日龄五指山猪心脏、肝脏、肺脏、脾脏、肾脏、肌肉、胃脏和小肠等8种组织中的mRNA表达水平.结果表明:(1)IGF2基因在8种组织中的表达呈相似的变化规律:各组织mRNA表达量随着日龄的增加而降低,30日龄时表达量最高,150龄时表达量最低,差异显著(P<0.01);(2)IGFBP3基因在心脏、肺、脾脏、肾脏、肠、胃等组织中的mRNA表达规律相似:即从30～150日龄,依次递减;而肝脏和肌肉mRNA表达从30日龄开始上升,肝脏在90日龄达最高,肌肉在60日龄时达最高,随后二者开始下降,在150日龄时达最低水平.以上结果初步揭示了五指山猪IGF2和IGFBP3基因表达的发育性变化模式,为深入研究五指山猪生长发育及矮小性状形成机理提供了基础数据.     

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.     

民猪和大白猪不同肌球蛋白重链表达差异

为揭示民猪与大白猪肌纤维类型组成差异,试验应用Real-time PCR法检测了民猪和大白猪背最长肌中肌球蛋白重链(MyHC)Ⅰ、Ⅱa、Ⅱx、Ⅱb 4种亚型mRNA表达差异。结果表明:同日龄民猪和大白猪、同体重民猪和大白猪相比,民猪Ⅰ、Ⅱa、Ⅱx型MyHC mRNA相对表达量显著高于大白猪(P0.05),而Ⅱb型MyHC mRNA相对表达量显著低于大白猪(P0.05)。说明民猪肌肉中氧化型和中间型肌纤维含量高于大白猪,而酵解型肌纤维含量低于大白猪。     

不同日龄东北民猪与大白猪背最长肌游离水和肌内脂肪含量的测定

研究测定了60、120、150、180、210日龄东北民猪与大白猪背最长肌游离水和肌内脂肪的含量,结果显示,120日龄大白猪背最长肌游离水含量极显著高于东北民猪1.25个百分点(P0.01),而210日龄东北民猪极显著高于大白猪2.27个百分点(P0.01),其他阶段无显著差异;60日龄东北民猪肌内脂肪含量极显著高于大白猪1.08个百分点(P0.01),180日龄东北民猪肌内脂肪含量显著高于大白猪0.94个百分点(P0.05),其他日龄无显著差异。上述结果表明,东北民猪背最长肌内游离水含量随着机体生长逐渐接近并超过大白猪,而大白猪背最长肌肌内脂肪沉积开始较晚,随着机体生长肌内脂肪含量逐渐接近东北民猪。     

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(P0.05),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(P0.01)。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(P0.01),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。     

绍鸭性成熟过程中血清IGF-I和leptin浓度的变化

试验采用放射免疫学方法（RIA）测定了绍鸭性成熟过程中血清leptin和IGF-I浓度的变化.结果显示,性成熟前30 d（90日龄）时血清leptin浓度显著升高,此后呈下降趋势;在1～150日龄期间,血清IGF-I浓度出现两个峰值,即在发育早期（30日龄）和性成熟（120日龄）时.以上结果显示：leptin和IGF-I可能是性成熟启动的两个代谢相关激素信号.     

沙子岭猪ASB17基因克隆、生物信息学分析及组织差异表达研究

本研究利用RACE法克隆沙子岭猪ASB17基因cDNA全长并进行生物信息学分析,应用实时荧光定量PCR对ASB17基因在沙子岭猪D30时期9个不同组织（睾丸、肌肉、小肠、肺脏、心脏、肾脏、肝脏、脾脏、脂肪）及睾丸组织8个不同发育阶段（E90、D1、D30、D60、D90、D120、D150、D180）进行差异表达分析。结果显示,ASB17基因克隆所得片段长1 135 bp,其中ORF区为888 bp,编码295个氨基酸。实时荧光定量PCR结果显示沙子岭猪D30时期ASB17 mRNA在睾丸中表达量最高,其次为脂肪及脾脏,而在肌肉及小肠中表达量最低,其中睾丸与肌肉、小肠及肺脏中表达量差异显著（P<0.05）;睾丸组织不同发育时期中,ASB17 mRNA在D120、D150时期表达量达到最高,在D180时期表达量略有下降,其中E90、D1、D30、D60与D90、D120、D150、D180时期差异极显著（P<0.01）;D90与D180时期差异不显著（P>0.05）,与其余时期均差异极显著（P<0.01）;沙子岭猪性成熟期D120、D150、D180 3个时期表达量差异不显著（P>0.05）。本试验成功克隆了沙子岭猪ASB17基因cDNA全长并检测了其在猪不同组织及睾丸组织不同发育阶段表达量,为进一步研究ASB17基因功能奠定基础。     

Evaluations of Boar Gonad Development,Spermatogenesis with regard to Semen Characteristics,Libido and Serum Testosterone Levels based on large White Duroc × Chinese Erhualian Crossbred Boars

Chinese Erhualian pigs are known for prolificacy with distinct reproductive traits compared with Western commercial breeds. In this study, a four‐generation intercross resource population was constructed using White Duroc boars and Chinese Erhualian sows as founder animals, and a total of 14 male reproductive traits were recorded in 411 F₂/F₃ boars including the testis and epididymis weights, the seminiferous tubular diameter and spermatogenesis at 60, 90 and 300 days of age, semen characteristics, serum testosterone concentration and libido level at 300 days of age. The White Duroc–Erhualian boars showed remarkable segregations in the traits measured except for the seminiferous tubular diameter and had high ratio (13.9%) of the abnormality of spermatogenesis, providing a good experimental population for detecting quantitative trait loci affecting these male reproductive traits. Furthermore, the correlations among nine male reproductive traits at 300 days of age indicated that the testis weight and the body weight were strongly correlated with the sperm production, supporting the two traits as important parameters for boar selection to increase sperm production and ultimately improve boar fertility. The libido level in the White Duroc–Erhualian boars that was evaluated by a new and easily recorded scoring system showed a significant correlation with serum testosterone concentration. Yet, both libido and serum testosterone concentration were not correlated with the sperm production. Results of this study provided new information on the male reproductive physiology and genetics in Chinese Erhualian and White Duroc boars.     

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.     

Cloning,Bioinfornatics and Tissue Differential Expression Analysis of ASB17 Gene in Shaziling Pig

In this study,ASB17 gene cDNA full length in Shaziling pig was cloned by RACE and it was analyzed by bioinformatics,the ASB17 gene expression levels were detected in Shaziling pig D30 period of nine different tissues（testis,muscle,intestine,lung,heart,kidney,liver,spleen,fat）and eight different developmental stages（E90,D1,D30,D60,D90,D120,D150,D180）of testicular tissue by Real-time quantitative PCR.The results showed that the length of ASB17 gene cDNA was 1 135 bp,and the ORF region was 888 bp,which encoded 295 amino acids.Real-time quantitative PCR results showed that the D30 period's expression level of ASB17 gene mRNA was the highest in testis,followed by fat and spleen,while the expression of ASB17 gene mRNA in muscle and small intestine tissue were the lowest,there were significant difference in testis and muscle,small intestine,lung（P<0.05);In the testis,the expression of ASB17 gene mRNA was the highest in D120 and D150 periods,while slightly declined in D180,the differences were extremely significant beween E90,D1,D30,D60 and D90,D120,D150,D180（P<0.01);The differences were not significant beween D90 and D180（P>0.05),while there were extremely significant with other periods（P<0.01);The sexual maturity periods of D120,D150,D180 had no significant differences（P>0.05).The full length of ASB17 cDNA was cloned and the expression levels of ASB17 gene in different tissues and different developmental stages were detected in this study,which laid the foundation for further research on the function of ASB17 gene.     

Study on Developmental Expression of ApoCⅡ Gene mRNA in Liver Tissues of Pigs

The aim of this study was to investigate the developmental patterns of ApoCⅡ gene mRNA in liver in Mashen and Large White pigs, and study the relationship between the expression level of ApoCⅡ and the lipid metabolism in pigs.The mRNA relative expressions of ApoCⅡ gene in liver at seven stages of 1,30,60,90,120,150,and 180-day old in Mashen and Large White pigs were determined by quantitative Real-time PCR.The results showed that the developmental trend of ApoCⅡ mRNA expression in liver between Mashen and Large White pigs was different.The ApoCⅡ mRNA abundance was decreased from birth to 60-day old,then increased at 90-day old,and decreased again after that in Mashen pig.However,the relative expression amount in Large White pig was gradually decreased from birth to 150-day old and increased again at 180-day old.Except for the ApoCⅡ mRNA expression amount at 1-day old,the differences of the expression amount at other stages in Mashen and Large White pigs were significant or extremely significant (P<0.05 or P<0.01).The ApoCⅡ mRNA expression in liver was affected by age and breed,and could play an important role in lipid metabolism in pigs.     

Relationships among measures of testicular development and endocrine function in boars

Nine blood samples were taken at 30-min intervals from 36 Landrace X Large White boars at each of eight ages (42, 56, 70, 84, 98, 112, 126 and 140 d). Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and estradiol-17 beta (E2) were quantified by radioimmunoassay procedures. The maximum concentration of LH and the age at maximum concentration were predicted for each boar. Variability of LH samples was described for each boar by the pooled within age variance among LH samples and by the number of LH peaks. Measurements of testicular development taken at 140 d of age included: in situ testis width and length, excised testis weights and histological traits of excised testes (seminiferous tubule diameter, percentage of tubules with a lumen and percentage of tubules with active spermatogenesis). Pooled within line correlations were calculated with data from boars selected for either high or low testis weight. Correlations among the testicular traits ranged from .45 to .88. Luteinizing hormone concentration (mean over all ages) was related to measures of testicular development (r = .24 to .49). Concentrations of LH from 42 to 84 d of age were more highly correlated with testicular traits than were the concentrations from 98 to 140 d.(ABSTRACT TRUNCATED AT 250 WORDS)     

11个猪种SLC6A14基因3个SNPs的群体遗传变异研究

本研究旨在了解猪种的遗传变异、种群间的亲缘关系和遗传分化。以白色杜洛克×二花脸资源家系F0代的17头二花脸母猪和2头白色杜洛克公猪的DNA池为模板,通过直接测序在猪SLC6A14基因内识别4个SNPs,以3个突变位点(g.7944AT、c.1438GA、g.21063GT)为基础,通过PCR-RFLP技术,对11个中外猪种进行多态性检测。结果表明:在SLC6A14g.7944AT和g.21063GT2个突变位点上,所有检测猪种都出现变异,其中槐猪、二花脸猪在g.7944AT位点上变异显著(0.01P0.05);而里岔黑猪、槐猪、玉山黑猪、合作藏猪、八眉猪在g.21063GT内变异极显著(P0.01),以GT基因型为主,且二花脸猪中出现TT基因型;在SLC6A14c.1438GA突变位点上,3个欧洲猪种(长白、大白、杜洛克)没有出现变异,均为GG纯合型,而其他猪种在此位点上均存在变异;再根据11个猪种3个位点的多态性信息计算Nei氏遗传距离,由UPGMA法构建聚类图,说明中国地方猪种与外引猪种存在明显的遗传分化;由此得出中国地方猪种比外引猪种有高的遗传多样性和变异,且存在明显的遗传分化,品种间多数猪种符合品种地域分布和品种特性。     

A genome scan for quantitative trait loci affecting male reproductive traits in a White Duroc x Chinese Erhualian resource population

Chinese Erhualian boars have dramatically smaller testes, greater concentrations of circulating androgens, and fewer Sertoli cells than Western commercial breeds. To identify QTL for boar reproductive traits, testicular weight, epididymal weight, seminiferous tubular diameter at 90 and 300 d, and serum testosterone concentration at 300 d were measured in 347 F(2) boars from a White Duroc x Chinese Erhualian cross. A whole genome scan was performed with 183 microsatellites covering 19 porcine chromosomes. A total of 16 QTL were identified on 9 chromosomes, including 1% genome-wide significant QTL for testicular weight at 90 and 300 d and seminiferous tubular diameter at 90 d on SSCX, and for epididymal weight and testosterone concentration at 300 d on SSC7. Two 5% genome-wide significant QTL were detected for testicular weight at 300 d on SSC1 and seminiferous tubular diameter at 300 d on SSC16. Nine suggestive QTL were found on SSC1, 2, 3, 5, 7, 13, and 14. Chinese Erhualian alleles were not systematically favorable for greater reproductive performance. This study confirmed the previous significant QTL for testicular weight on SSCX and for epididymal weight on SSC7, and reported QTL for seminiferous tubular diameter and testosterone concentration at the first time. The observed different QTL for the same trait at different ages reflect the involvement of distinct genes in the development of male reproductive traits.