本文選題：新西蘭肉兔　切入點(diǎn)：PID1基因　出處：《山東農(nóng)業(yè)大學(xué)》2014年碩士論文

【摘要】：隨著人們生活水平的不斷提高，豬肉的品質(zhì)也隨之受到人們?cè)絹?lái)越多的關(guān)注。眾多肉品質(zhì)性狀之中，肌內(nèi)脂肪（IMF）是衡量豬肉品質(zhì)的一個(gè)非常重要的指標(biāo)，其含量與豬肉的多汁性、嫩度以及風(fēng)味密切相關(guān)。有研究發(fā)現(xiàn)，肥胖人群脂肪細(xì)胞的增殖與PID1(磷酸酪氨酸互作結(jié)構(gòu)域1)基因密切相關(guān)，，在肥胖人群中呈高豐度表達(dá)。本實(shí)驗(yàn)室前期在萊蕪豬、魯萊黑豬、大白豬三個(gè)豬種上對(duì)PID1基因的研究提示，肌內(nèi)脂肪的含量與PID1基因密切相關(guān)。為進(jìn)一步研究PID1基因的功能，本實(shí)驗(yàn)在前期成功構(gòu)建并篩選得到的高效PID1基因RNA干擾表達(dá)載體pGPU6/GFP/Neo-PID1-2的基礎(chǔ)上，利用精子介導(dǎo)法制備RNA干擾PID1轉(zhuǎn)基因肉兔模型，驗(yàn)證PID1基因?qū)?nèi)脂肪沉積的影響，并以萊蕪豬和魯萊黑豬為實(shí)驗(yàn)材料，研究了PID1基因的多態(tài)性與肌內(nèi)脂肪含量的關(guān)系。主要研究結(jié)果如下： 一、利用前期成功構(gòu)建的高效干擾表達(dá)載體pGPU6/GFP/Neo-PID1-2與Roche的X-treme GENE HP轉(zhuǎn)染試劑共孵育15min后，加入適當(dāng)?shù)腄MEM配成注射液。將公兔麻醉后，對(duì)實(shí)驗(yàn)組公兔兩側(cè)睪丸進(jìn)行多點(diǎn)注射，對(duì)照組注射等量生理鹽水。每周注射一次，共注射三次。在相同且適宜的條件下飼養(yǎng)30天后，試驗(yàn)組和對(duì)照組分別按照公母比例1:6和1:4進(jìn)行配種，獲得F1代個(gè)體。 二、在轉(zhuǎn)基因后代中，妊娠期結(jié)束試驗(yàn)組共分娩活仔兔106只，其中陽(yáng)性兔7只，陰性兔99只，陽(yáng)性率為6.60%。對(duì)繁殖的F1代肉兔個(gè)體進(jìn)行活體熒光檢測(cè)、PCR檢測(cè)，并對(duì)不同組別（陽(yáng)性組、陰性組、空白對(duì)照組）體重達(dá)3.0kg左右的F1代肉兔抽樣屠宰對(duì)其背最長(zhǎng)肌進(jìn)行肌內(nèi)脂肪的測(cè)定，并進(jìn)行qRT-PCR和Western blot驗(yàn)證。在F1代中，試驗(yàn)組轉(zhuǎn)基因陽(yáng)性兔的肌內(nèi)脂肪含量顯著低于陰性兔和空白對(duì)照兔（P0.05），qRT-PCR和Western blot檢測(cè)結(jié)果也顯示陽(yáng)性組PID1基因mRNA和蛋白表達(dá)量顯著降低；同時(shí)也對(duì)各組F1代肉兔進(jìn)行了日增重、全凈膛率、半凈膛率、肌肉滴水損失、嫩度、屠宰后45min pH、24h pH、失水率以及24h SOD、T-AOC、MDA等指標(biāo)的檢測(cè)，結(jié)果表明轉(zhuǎn)基因處理對(duì)日增重、凈膛率、滴水損失、嫩度、24h pH有顯著影響。 三、以萊蕪豬和魯萊黑豬為實(shí)驗(yàn)材料（萊蕪豬67頭，魯萊黑豬40頭），混池測(cè)序發(fā)現(xiàn)，在PID1基因CDS區(qū)的第267位點(diǎn)發(fā)生了堿基C→T的突變，SSCP分析發(fā)現(xiàn)，在兩個(gè)品種中，CC型個(gè)體肌內(nèi)脂肪含量為9.77%±5.03%，CT型的肌內(nèi)脂肪含量為6.11%±1.89%，TT型的肌內(nèi)脂肪含量為5.71%±2.59%。在兩個(gè)群體中，C為優(yōu)勢(shì)等位基因，基因頻率為0.56；在萊蕪豬群體中，CC型個(gè)體的肌內(nèi)脂肪含量為10.38%±5.25%，CT型的肌內(nèi)脂肪含量為6.38%±2.03%，TT型的肌內(nèi)脂肪含量為6.03%±2.39%，C基因頻率為0.6。在魯萊黑豬群體中，CC型的肌內(nèi)脂肪含量為7.37%±5.77%，CT型的肌內(nèi)脂肪含量為5.15%±1.13%，TT型的肌內(nèi)脂肪含量為4.74%±2.73%，群體中各類基因型分布均達(dá)到哈代—溫伯格平衡狀態(tài)。 綜上表明，PID1基因與肌內(nèi)脂肪沉積密切相關(guān)。本研究從RNAi肉兔模型構(gòu)建的角度進(jìn)一步驗(yàn)證了PID1基因?qū)?nèi)脂肪沉積影響的功能，也為下一步研究制備高肌內(nèi)脂肪優(yōu)質(zhì)PID1轉(zhuǎn)基因豬奠定了基礎(chǔ)。此外，轉(zhuǎn)基因處理后對(duì)其他一些肉質(zhì)性狀也存在一定的影響；本研究發(fā)現(xiàn)的突變位點(diǎn)可能與肌內(nèi)脂肪沉積存在聯(lián)系，還需進(jìn)一步驗(yàn)證。
[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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本文編號(hào)：1666347