【摘要】：極低密度脂蛋白受體(very low density lipoprotein receptor,VLDLR)是低密度脂蛋白受體家族中的一員,主要作用是通過與富含ApoE的脂蛋白結(jié)合來調(diào)控甘油三酯的代謝。在動物體內(nèi),VLDLR主要分布于骨骼肌、脂肪、心臟等組織,在肝臟中分布較少。本實驗在前期實驗的基礎(chǔ)上,對高郵鴨VLDLR基因的多態(tài)性進行了進一步的分析,在5'端UTR及信號肽編碼區(qū)位置檢測出4出個新的SNPs位點,同時對VLDLR基因多態(tài)性與高郵鴨生長性狀和屠宰性狀進行了相關(guān)分析;在通過RT-PCR的方法對正常鴨和雞不同組織VLDLR mRNA的表達分布情況進行研究的基礎(chǔ)上,對MD雞不同組織VLDLR mRNA的表達進行檢測,以期研究發(fā)生腫瘤的禽組織VLDLR mRNA的表達變化情況。1高郵鴨VLDLR基因5'端UTR及信號肽編碼區(qū)多態(tài)性與生長和屠宰性狀的關(guān)聯(lián)分析本實驗以高郵鴨群體為研究對象,采用PCR擴增、測序法等方法測定了267個樣VLDLR基因5'端UTR及信號肽編碼區(qū)部分序列,在該段序列中檢測出4個SNPs(g.151GA、g.170CT、g.206AG、g.278-295+-),基于這4個SNPs位點,采用PHASE2.0軟件構(gòu)建了8種單倍型,其中ACG+為主要單倍型,頻率高達29.81%;GTG+和ACA+單倍型頻率最低,均為0.96%。另外,在8個單倍型的基礎(chǔ)上構(gòu)建了15種雙倍型,其中H4H8頻率最高,為28.16%。關(guān)聯(lián)分析結(jié)果表明,在第6周齡,雙倍型H1H1的體重顯著高于H2H8型(P0.05);在7至10周齡,雙倍型H1H1的體重極顯著高于H2H8型(P0.01),顯著高于H4H8型(P0.05)。與此同時,雙倍型H5H8的腹脂率極顯著高于H1H1型和H2H8型(P0.01),顯著高于H4H8型(P0.05),雙倍型H4H8的腹脂率顯著高于H1H1型(P0.05)。最小二乘法分析結(jié)果表明,雙倍型H1H1的平均體重最高,H2H8最低;雙倍型H2H8的屠宰率、半凈膛率和全凈膛率最高;雙倍型H5H8的腹脂率最高,H4H8型次之,H1H1型最低。表明雙倍型H1H1為優(yōu)勢組合,有可能用來作為提高高郵鴨胴體性能和選擇高瘦肉率優(yōu)良品種的分子標記。2家禽VLDLR基因mRNA在不同組織中的表達分析為研究VLDLR在腫瘤發(fā)生中的表達變化,本實驗首先對VLDLR基因在鴨和雞不同組織內(nèi)的表達水平和分布情況進行探討�？焖俨杉哙]鴨和成年海蘭白雞骨骼肌、肺臟、脂肪、脾臟、肝臟5種組織提取RNA,反轉(zhuǎn)錄成cDNA保存于-20℃?zhèn)溆�。根�?jù)GenBank中的鴨和雞VLDLR基因序列設(shè)計引物并進行RT-PCR擴增,檢測5種組織中VLDLR基因的相對表達水平。結(jié)果顯示VLDLR基因在鴨和雞不同組織間的表達存在差異,均表現(xiàn)為骨骼肌表達量最高,肺臟和脂肪組織次之,肝臟表達量最低。鴨的5種組織相對表達量表現(xiàn)為為骨骼肌(57.47±0.10)肺臟(11.72±0.28)脂肪組織(4.13±0.05)脾臟(1.76±0.14)肝臟(i.00±0.15);雞表現(xiàn)為骨骼肌(10.95±0.14)肺臟(3.87±0.23)脂肪組織(3.02士0.11)脾臟(2.31±0.08)肝臟(1.00±0.17)。二者相同,脾臟、肺臟、骨骼肌和脂肪內(nèi)VLDLRmRNA的表達均極顯著高于肝臟(P0.01),肺臟內(nèi)VLDLRmRNA的表達高于脂肪,二者差異極顯著(P0.01)。本實驗顯示了 VLDLR基因在肺臟有較高的表達量,脾臟也有一定的表達。3 MDV對雞VLDLR mRNA表達的影響本實驗研究對象為自然條件下感染MDV雞、MDV RB1B毒株攻毒雞和禽霍亂(PM)雞。采集3種雞肝臟、脾臟、肺臟、骨骼肌、脂肪等組織提取RNA,反轉(zhuǎn)錄成cDNA于-20℃保存?zhèn)溆�。根�?jù)GenBank中的雞VLDLR基因序列設(shè)計引物并進行RT-PCR擴增,檢測雞VLDLR mRNA在5個組織中的相對表達水平。結(jié)果顯示VLDLR基因在3種雞不同組織間的相對表達存在差異。在感染MDV雞不同組織內(nèi)的相對表達情況為骨骼肌(7.88±0.095)肝臟(1.00±0.041)肺(0.649±0.027)脂肪(0.168±0.053)脾臟(0.05±0.021),脾臟、肺臟和脂肪組織內(nèi)VLDLRmRNA的表達量均極顯著低于肝臟的表達量(P0.01),同時VLDLR在肺臟的表達量比脂肪組織高,表現(xiàn)為差異極顯著(P0.01)。在攻毒MDV RB1B毒株雞不同組織的相對表達情況為骨骼肌(30.18±0.074)脂肪(5.13±0.061)肺(2.99±0.178)肝臟(1.00±0.390)脾臟(0.41±0.117),脾臟內(nèi) VLDLR mRNA的表達量低于肝臟,且兩者差異不顯著,肺臟、骨骼肌和脂肪組織內(nèi)VLDLR表達量均顯著高于肝臟(P0.01),脂肪內(nèi)VLDLRmRNA的表達高于肺,二者差異極顯著(P0.01)。在PM雞不同組織的相對表達情況為骨骼肌(84.83±0.183)肺(13.17±0.307)脂肪(2.72±0.050)脾臟(1.17±0.214)肝臟(1.00±0.343),脾臟的表達量略高于肝臟,二者差異不顯著,肺臟、骨骼肌和脂肪內(nèi)VLDLRrmRNA的表達極顯著高于肝臟(P0.01),肺臟內(nèi)VLDLRmRNA的表達高于脂肪,二者差異極顯著(P0.01)。結(jié)果顯示,MDV感染雞肝臟(發(fā)生腫瘤的肝臟)組織中VLDLRmRNA的表達超過了脂肪、肺臟等組織,而攻毒MDV RB1B毒株后未發(fā)生MDV感染或被其他疾病感染(如PM)的肝臟內(nèi)VLDLRmRNA的表達量依然較低。
[Abstract]:Very low density lipoprotein receptor (VLDLR) is a member of the low density lipoprotein receptor family. Its main function is to regulate the metabolism of triglycerides by binding to apoE-rich lipoproteins. On the basis of previous experiments, the polymorphisms of VLDLR gene in Gaoyou duck were further analyzed. Four new SNPs were detected at 5'UTR and signal peptide coding region. The correlation between VLDLR gene polymorphisms and growth traits and slaughter traits of Gaoyou duck was analyzed. The normal ducks and Gaoyou duck were analyzed by RT-PCR. On the basis of the study on the expression and distribution of VLDLR mRNA in different tissues of chickens, the expression of VLDLR mRNA in different tissues of MD chickens was detected in order to study the changes of VLDLR mRNA expression in tumorigenic poultry tissues. 1 Correlation analysis of 5'UTR and signal peptide coding region polymorphism of VLDLR gene with growth and slaughter traits in Gaoyou duck Based on the four SNPs, eight haplotypes were constructed by PHASE 2.0 software, in which ACG + was the dominant factor. Four SNPs (g.151GA, g.170CT, g.206AG, g.278-295+-) were detected in 267 samples of Gaoyou duck population. The haplotype frequency was as high as 29.81%; GTG + and ACA + haplotypes had the lowest frequency (0.96%). In addition, 15 haplotypes were constructed on the basis of eight haplotypes, of which H4H8 frequency was the highest (28.16%). At the same time, the abdominal fat rate of diploid H5H8 was significantly higher than that of H1H1 and H2H8 (P 0.01), significantly higher than that of H4H8 (P 0.05), and the abdominal fat rate of diploid H4H8 was significantly higher than that of H1H1 (P 0.05). H8 had the highest carcass percentage, semi-clean rate and full-clean rate, double H5H8 had the highest abdominal fat percentage, H4H8 had the second highest abdominal fat percentage, H1H1 had the lowest abdominal fat percentage, indicating that double H1H1 was the dominant combination and could be used as a molecular marker for improving carcass performance and selecting high lean meat percentage of Gaoyou duck. To study the changes of VLDLR expression in tumorigenesis, the expression level and distribution of VLDLR gene in different tissues of ducks and chickens were investigated. RNA was extracted from skeletal muscle, lung, fat, spleen and liver of Gaoyou ducks and adult Hailan white chickens, and then retranscribed into cDNA and stored at - 20 C for reserve according to GenBank. The relative expression of VLDLR gene in duck and chicken tissues was detected by RT-PCR. The results showed that the expression of VLDLR gene was different between duck and chicken tissues. The expression of VLDLR gene was highest in skeletal muscle, followed by lung and adipose tissue, and lowest in liver. The expression was skeletal muscle (57.47 [(57.47 [0.10) lung (11.72 [(11.72 [0.28), (11.72 [(11.72 [0.28), (4.13 [0.05] (1.76 [0.14] (i.00 [0.15); (chickchicken (10.95 [(10.95 [0.95 [0.14]) lung (3.87 [0.23] adipose tissue (3.02 [0.02] 0.11] sple (2.31 [0.08] liver (1.00 [0.00] 17). Both were the same, sple, sple, lung fat, lung, lung fat, lung Watch The expression of VLDLR mRNA in lung was significantly higher than that in liver (P 0.01), and the expression of VLDLR mRNA in lung was significantly higher than that in fat (P 0.01). Virus chickens and poultry cholera (PM) chickens. RNA was extracted from liver, spleen, lung, skeletal muscle, fat and other tissues of three kinds of chickens, and then retranscribed into cDNA for preservation at - 20 C. Primers were designed according to the chicken VLDLR gene sequence in GenBank and RT-PCR was used to detect the relative expression level of VLDLR mRNA in five tissues. The expression of VLDLR mRNA in different tissues of MDV-infected chickens was significantly lower than that in the liver (P 0.01), and the expression of VLDLR mRNA in skeletal muscle (7.88.095) liver (1.00.041) lung (0.649.027) fat (0.168.053) spleen (0.05.021). The relative expression of VLDLR mRNA in different tissues of chickens infected with MDV RB1B strain was skeletal muscle (30.18.074) fat (5.13.061) lung (2.99.178) liver (1.00.390) spleen (0.41.117), and there was no significant difference between them. 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 significantly higher than that in lung (P 0.01). The relative expression of VLDLR mRNA in different tissues of PM chicken was skeletal muscle (84.83.183) lung (13.17.307) fat (2.72.050) spleen (1.17.214) liver (1.00.343), spleen (1.00.343). The expression of VLDLR mRNA in lung, skeletal muscle and fat was significantly higher than that in liver (P 0.01), and the expression of VLDLR mRNA in lung was significantly higher than that in fat (P 0.01). The results showed that the expression of VLDLR mRNA in MDV infected chicken liver (tumorigenic liver) was higher than that in fat, lung and so on. However, the expression of VLDLR mRNA in the liver of patients who had not been infected by MDV or had been infected by other diseases (such as PM) was still low.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S83

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