【作者】 王聪；

【导师】 张汤杰； 宋卫涛；

【作者基本信息】 扬州大学 ， 兽医硕士（专业学位）， 2017， 硕士

【摘要】 极低密度脂蛋白受体(very low density lipoprotein receptor,VLDLR)是低密度脂蛋白受体家族中的一员,主要作用是通过与富含ApoE的脂蛋白结合来调控甘油三酯的代谢。在动物体内,VLDLR主要分布于骨骼肌、脂肪、心脏等组织,在肝脏中分布较少。本实验在前期实验的基础上,对高邮鸭VLDLR基因的多态性进行了进一步的分析,在5’端UTR及信号肽编码区位置检测出4出个新的SNPs位点,同时对VLDLR基因多态性与高邮鸭生长性状和屠宰性状进行了相关分析;在通过RT-PCR的方法对正常鸭和鸡不同组织VLDLR mRNA的表达分布情况进行研究的基础上,对MD鸡不同组织VLDLR mRNA的表达进行检测,以期研究发生肿瘤的禽组织VLDLR mRNA的表达变化情况。1高邮鸭VLDLR基因5’端UTR及信号肽编码区多态性与生长和屠宰性状的关联分析本实验以高邮鸭群体为研究对象,采用PCR扩增、测序法等方法测定了267个样VLDLR基因5’端UTR及信号肽编码区部分序列,在该段序列中检测出4个SNPs(g.151G>A、g.170C>T、g.206A>G、g.278-295+>-),基于这4个SNPs位点,采用PHASE2.0软件构建了8种单倍型,其中ACG+为主要单倍型,频率高达29.81%;GTG+和ACA+单倍型频率最低,均为0.96%。另外,在8个单倍型的基础上构建了15种双倍型,其中H4H8频率最高,为28.16%。关联分析结果表明,在第6周龄,双倍型H1H1的体重显著高于H2H8型(P<0.05);在7至10周龄,双倍型H1H1的体重极显著高于H2H8型(P<0.01),显著高于H4H8型(P<0.05)。与此同时,双倍型H5H8的腹脂率极显著高于H1H1型和H2H8型(P<0.01),显著高于H4H8型(P<0.05),双倍型H4H8的腹脂率显著高于H1H1型(P<0.05)。最小二乘法分析结果表明,双倍型H1H1的平均体重最高,H2H8最低;双倍型H2H8的屠宰率、半净膛率和全净膛率最高;双倍型H5H8的腹脂率最高,H4H8型次之,H1H1型最低。表明双倍型H1H1为优势组合,有可能用来作为提高高邮鸭胴体性能和选择高瘦肉率优良品种的分子标记。2家禽VLDLR基因mRNA在不同组织中的表达分析为研究VLDLR在肿瘤发生中的表达变化,本实验首先对VLDLR基因在鸭和鸡不同组织内的表达水平和分布情况进行探讨。快速采集高邮鸭和成年海兰白鸡骨骼肌、肺脏、脂肪、脾脏、肝脏5种组织提取RNA,反转录成cDNA保存于-20℃备用。根据GenBank中的鸭和鸡VLDLR基因序列设计引物并进行RT-PCR扩增,检测5种组织中VLDLR基因的相对表达水平。结果显示VLDLR基因在鸭和鸡不同组织间的表达存在差异,均表现为骨骼肌表达量最高,肺脏和脂肪组织次之,肝脏表达量最低。鸭的5种组织相对表达量表现为为骨骼肌(57.47±0.10)>肺脏(11.72±0.28)>脂肪组织(4.13±0.05)>脾脏(1.76±0.14)>肝脏(i.00±0.15);鸡表现为骨骼肌(10.95±0.14)>肺脏(3.87±0.23)>脂肪组织(3.02士0.11)>脾脏(2.31±0.08)>肝脏(1.00±0.17)。二者相同,脾脏、肺脏、骨骼肌和脂肪内VLDLRmRNA的表达均极显著高于肝脏(P<0.01),肺脏内VLDLRmRNA的表达高于脂肪,二者差异极显著(P<0.01)。本实验显示了 VLDLR基因在肺脏有较高的表达量,脾脏也有一定的表达。3 MDV对鸡VLDLR mRNA表达的影响本实验研究对象为自然条件下感染MDV鸡、MDV RB1B毒株攻毒鸡和禽霍乱(PM)鸡。采集3种鸡肝脏、脾脏、肺脏、骨骼肌、脂肪等组织提取RNA,反转录成cDNA于-20℃保存备用。根据GenBank中的鸡VLDLR基因序列设计引物并进行RT-PCR扩增,检测鸡VLDLR mRNA在5个组织中的相对表达水平。结果显示VLDLR基因在3种鸡不同组织间的相对表达存在差异。在感染MDV鸡不同组织内的相对表达情况为骨骼肌(7.88±0.095)>肝脏(1.00±0.041)>肺(0.649±0.027)>脂肪(0.168±0.053)>脾脏(0.05±0.021),脾脏、肺脏和脂肪组织内VLDLRmRNA的表达量均极显著低于肝脏的表达量(P<0.01),同时VLDLR在肺脏的表达量比脂肪组织高,表现为差异极显著(P<0.01)。在攻毒MDV RB1B毒株鸡不同组织的相对表达情况为骨骼肌(30.18±0.074)>脂肪(5.13±0.061)>肺(2.99±0.178)>肝脏(1.00±0.390)>脾脏(0.41±0.117),脾脏内 VLDLR mRNA的表达量低于肝脏,且两者差异不显著,肺脏、骨骼肌和脂肪组织内VLDLR表达量均显著高于肝脏(P<0.01),脂肪内VLDLRmRNA的表达高于肺,二者差异极显著(P<0.01)。在PM鸡不同组织的相对表达情况为骨骼肌(84.83±0.183)>肺(13.17±0.307)>脂肪(2.72±0.050)>脾脏(1.17±0.214)>肝脏(1.00±0.343),脾脏的表达量略高于肝脏,二者差异不显著,肺脏、骨骼肌和脂肪内VLDLRrmRNA的表达极显著高于肝脏(P<0.01),肺脏内VLDLRmRNA的表达高于脂肪,二者差异极显著(P<0.01)。结果显示,MDV感染鸡肝脏(发生肿瘤的肝脏)组织中VLDLRmRNA的表达超过了脂肪、肺脏等组织,而攻毒MDV RB1B毒株后未发生MDV感染或被其他疾病感染(如PM)的肝脏内VLDLRmRNA的表达量依然较低。更多还原

【Abstract】 The very low density lipoprotein receptor(VLDLR)is a member of the low density lipoprotein receptor family.This receptor has an important role in cholesterol uptake,metabolism of apoprotein-E-containing triacylglycerol-rich lipoproteins the main function is to regulate the metabolism of triglycerides by binding to ApoE rich lipoproteins.In animals,VLDLR is mainly distributed in skeletal muscle,fat,heart and other tissues but liver.On the basis of previous experiments,4 SNPs loci were screened by sequencing the VLDLR gene of Gaoyou duck to study on the relationship between genetic polymorphism and growth traits and carcass traits of Gaoyou ducks.The expression of VLDLR mRNA in different tissues of ducks and chickens was studied by RT-PCR.Finally,the expression of VLDLR mRNA in different tissues of MD was detected by RT-PCR for the study of the expression of VLDLR mRNA in different tumor tissues of poultry.1 Polymorphism of VLDLR signal peptide coding region and its association with growth and carcass traits in Gaoyou ducks.The purpose of this study was to investigate the relationship between VLDLR genetic polymorphism and body weight plus abdominal fat traits of Gaoyou ducks.A primer pairs for the 5’-UTR and signal peptide coding of the duck VLDLR gene was designed based on the duck genomic sequence database.Polymorphisms were detected by polymerase chain reaction(PCR)and DNA sequencing.Four novel SNPs(g.151G>A,g.170C>T,g.206A>G and g.278-295+>-)were detected in the 374 bp.Based on the four SNPs above,8 haplotypes were identified.The H8 was the most common haplotype in Gaoyou population occurring at a frequency of more than 31%.Haplotype combinations constructed on these SNPs were associated with body weight at 6-10 weeks of age(P<0.05,P<0.01)and abdominal fat percentage(AFP)(P<0.05,P<0.01).In particular,diplotype H1H1 had positive effect on body weight from 6th to 10th weeks of age,whereas it had negative effect on abdominal fat percentage(AFP).Positive effects of the diplotype H5H8 were observed for both AFP and body weight.This suggested that the VLDLR gene was playing an important role in the regulation of body weight and fat-related traits and could be a potential marker in a marker assisted selection program during duck breeding.2 Study on VLDLR mRNA Expression of Different Tissues in poultryFor the study of VLDLR expression in tumorigenesis,this study was to investigate the expression level and distribution of VLDLR gene in different tissues of ducks and chickens at first.Lung,fat,spleen,liver and other tissues were collected quickly and stored in liquid nitrogen for RNA extraction.The expression of VLDLR gene in five tissues of ducks and chickens was analyzed by designing primers based on VLDLR gene sequence in GenBank and performing RT-PCR amplification.The relative expression level of VLDLR gene in five tissues was detected.The results showed that the expression of VLDLR gene was different among different tissues of ducks and chickens.The expression of VLDLR gene was the highest in muscle,followed by lung and adipose tissue and lowest in liver.The relative expression were skeletal muscle(57.47±0.10)>lung(11.72±0.28)>Adipose tissue(4.13±0.05)>spleen(1.76 ± 0.14)>liver(1.00±0.15)in ducks and skeletal muscle(10.95±0.14)>lung(3.87±0.23)>Adipose tissue(3.02±0.11)>spleen(2.31±0.08)>liver(1.00±0.17)in chickens.Two identical,the expression of VLDLR mRNA in spleen,lung,skeletal muscle and fat was significantly higher than that in liver(P<0.01).The expression of VLDLR mRNA in lung was higher than that in fat,the difference between the two groups was very significant(P<0.01).This study showed firstly that VLDLR gene in the lungs have a higher expression,the spleen also has a certain expression.3 The effect of MDV on the mRNA expression of VLDLRThe animals of this study was chicken suspected infected MDV,poisoned MDV RB1B and suspected PM.Liver,spleen,lung,skeletal muscle and adipose tissue were collected quickly and stored in liquid nitrogen for RNA extraction.The expression of VLDLR gene in five tissues of three kinds of chickens was analyzed by designing primers based on VLDLR gene sequence in GenBank and performing RT-PCR amplification.The results showed that the expression of VLDLR gene was different among different tissues of three kinds of chickens.The relative expression were skeletal muscle(7.88±0.095)>liver(1.00±0.041)>lung(0.649±0.027)>adipose tissue(0.168±0.053)>spleen(0.05±0,021)in chicken infected MDV,the expression of VLDLR mRNA in spleen,lung,and adipose tissue was significantly lower than that in liver(P<0.01).At the same time,the expression of VLDLR in lung was higher than that in adipose tissue,and the difference was very significant(P<0.01).The relative expression of different tissues was skeletal muscle(30.18 ± 0.074)>adipose tissue(5.13±0.061)>lung(2.991±0.178)>liver(1.00±0.390)>spleen(0.41 ±0.117)in chickens poisoned MDV RB1B,the expression of VLDLR mRNA in the spleen was lower than that in the liver,and the difference was not significant,the expression of VLDLR in lung,skeletal muscle and adipose tissue was significantly higher than that in liver(P<0.01),the expression of VLDLR mRNA in adipose tissue was higher than that in lung,the difference between the two groups was very significant(P<0.01).The relative expression of different tissues was skeletal muscle(84.83±0.183)>lung(13.17±0.307)>adipose tissue(2.72±0.050)>spleen(1.17±0.214)>liver(1.00±0.343)in PM.The expression of the spleen was slightly higher than that of liver and no significant difference between the two groups,the expression of VLDLR mRNA in lung,skeletal muscle and adipose tissue was significantly higher than that of liver(P<0.01),the VLDLR mRNA expression in lung was higher than adipose tissue,and the difference was very significant(P<0.01).The resules showed that the expression of VLDLR mRNA in liver of chicken suspccted infected MDV(Tumor liver)rised and higher than adipose tissue,spleen and other tissues,the expression of VLDLR mRNA was still low in liver no tumor or infected by other diseases.更多还原