RNA干扰沉默PID1基因肉兔模型的构建及猪PID1基因多态性与肌内脂肪含量关联分析

发布时间：2018-03-26 04:33

  本文选题：新西兰肉兔　切入点：PID1基因　出处：《山东农业大学》2014年硕士论文

【摘要】：随着人们生活水平的不断提高，猪肉的品质也随之受到人们越来越多的关注。众多肉品质性状之中，肌内脂肪（IMF）是衡量猪肉品质的一个非常重要的指标，其含量与猪肉的多汁性、嫩度以及风味密切相关。有研究发现，肥胖人群脂肪细胞的增殖与PID1(磷酸酪氨酸互作结构域1)基因密切相关，，在肥胖人群中呈高丰度表达。本实验室前期在莱芜猪、鲁莱黑猪、大白猪三个猪种上对PID1基因的研究提示，肌内脂肪的含量与PID1基因密切相关。为进一步研究PID1基因的功能，本实验在前期成功构建并筛选得到的高效PID1基因RNA干扰表达载体pGPU6/GFP/Neo-PID1-2的基础上，利用精子介导法制备RNA干扰PID1转基因肉兔模型，验证PID1基因对肌内脂肪沉积的影响，并以莱芜猪和鲁莱黑猪为实验材料，研究了PID1基因的多态性与肌内脂肪含量的关系。主要研究结果如下： 一、利用前期成功构建的高效干扰表达载体pGPU6/GFP/Neo-PID1-2与Roche的X-treme GENE HP转染试剂共孵育15min后，加入适当的DMEM配成注射液。将公兔麻醉后，对实验组公兔两侧睾丸进行多点注射，对照组注射等量生理盐水。每周注射一次，共注射三次。在相同且适宜的条件下饲养30天后，试验组和对照组分别按照公母比例1:6和1:4进行配种，获得F1代个体。 二、在转基因后代中，妊娠期结束试验组共分娩活仔兔106只，其中阳性兔7只，阴性兔99只，阳性率为6.60%。对繁殖的F1代肉兔个体进行活体荧光检测、PCR检测，并对不同组别（阳性组、阴性组、空白对照组）体重达3.0kg左右的F1代肉兔抽样屠宰对其背最长肌进行肌内脂肪的测定，并进行qRT-PCR和Western blot验证。在F1代中，试验组转基因阳性兔的肌内脂肪含量显著低于阴性兔和空白对照兔（P0.05），qRT-PCR和Western blot检测结果也显示阳性组PID1基因mRNA和蛋白表达量显著降低；同时也对各组F1代肉兔进行了日增重、全净膛率、半净膛率、肌肉滴水损失、嫩度、屠宰后45min pH、24h pH、失水率以及24h SOD、T-AOC、MDA等指标的检测，结果表明转基因处理对日增重、净膛率、滴水损失、嫩度、24h pH有显著影响。 三、以莱芜猪和鲁莱黑猪为实验材料（莱芜猪67头，鲁莱黑猪40头），混池测序发现，在PID1基因CDS区的第267位点发生了碱基C→T的突变，SSCP分析发现，在两个品种中，CC型个体肌内脂肪含量为9.77%±5.03%，CT型的肌内脂肪含量为6.11%±1.89%，TT型的肌内脂肪含量为5.71%±2.59%。在两个群体中，C为优势等位基因，基因频率为0.56；在莱芜猪群体中，CC型个体的肌内脂肪含量为10.38%±5.25%，CT型的肌内脂肪含量为6.38%±2.03%，TT型的肌内脂肪含量为6.03%±2.39%，C基因频率为0.6。在鲁莱黑猪群体中，CC型的肌内脂肪含量为7.37%±5.77%，CT型的肌内脂肪含量为5.15%±1.13%，TT型的肌内脂肪含量为4.74%±2.73%，群体中各类基因型分布均达到哈代—温伯格平衡状态。 综上表明，PID1基因与肌内脂肪沉积密切相关。本研究从RNAi肉兔模型构建的角度进一步验证了PID1基因对肌内脂肪沉积影响的功能，也为下一步研究制备高肌内脂肪优质PID1转基因猪奠定了基础。此外，转基因处理后对其他一些肉质性状也存在一定的影响；本研究发现的突变位点可能与肌内脂肪沉积存在联系，还需进一步验证。
[Abstract]:With the continuous improvement of living standards, the quality of pork has also attracted more and more attention. Many traits of meat quality and intramuscular fat (IMF) is a very important index to measure the quality of pork, pork and its content of juiciness, tenderness and flavor are closely related. Studies have found that obesity and proliferation of PID1 cells (phosphotyrosine interacting domain 1) gene closely related in obese people showed high expression abundance. The laboratory in Laiwu lulai black pig, large white pig, three pigs of PID1 gene and PID1 gene suggest that the content of intramuscular fat closely related. To further study the function of PID1 gene, we constructed in the early success of efficient PID1 RNA gene and screened interference expression vector pGPU6/GFP/Neo-PID1-2, using RNA PID1 preparation of sperm mediated gene transfer mediated by interference The rabbit model to verify the effect of PID1 gene on intramuscular fat deposition, and to Laiwu and lulai pigs as experimental material, studied the relationship between fat content and PID1 gene polymorphism with intramuscular. The main results are as follows:
A high expression of interference constructed by the early success of the X-treme GENE transfection reagent HP vector pGPU6/GFP/Neo-PID1-2 and Roche were co incubated with 15min after adding DMEM into injection. Male rabbits were anesthetized and multi point injection on the experimental group of male rabbit testes, the control group injected saline injected once a week. Co injection, three times. After 30 days in the same and the suitable conditions, the test group and the control group respectively according to the proportion of male and female 1:6 and 1:4 hybridization, F1 generation individuals.
Two, in the transgenic progeny, pregnancy test group were alive over the delivery of 106 rabbits, which were 7 rabbits, 99 rabbits were negative, the positive rate was 6.60%. on the propagation of F1 generation in vivo rabbit individual fluorescence detection, PCR detection, and the different groups (positive group, negative group, blank control weight about 3.0kg group) F1 generation of rabbit longissimus muscle were slaughtered for sampling of intramuscular fat on the back, and qRT-PCR and Western. Blot verification in the F1 generation, the fat content of experimental group transgenic rabbit muscle was significantly lower than that of negative control rabbits and rabbits (P0.05), qRT-PCR and Western blot the test results also showed that the expression of PID1 gene and mRNA protein positive group was significantly reduced; but also for each generation of rabbit F1 ADG, eviscerated rate, slaughter rate, muscle drip loss, tenderness, 45min pH 24h pH after slaughter, water loss rate and 24h, SOD, T-AOC, MDA and other indicators The results showed that the transgenic treatment had a significant effect on the daily weight gain, the net chamber rate, the loss of drip water, the tenderness and the 24h pH.
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