本文選題：齊口裂腹魚(yú) + MHCⅡA��； 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：本文以齊口裂腹魚(yú)為研究對(duì)象,克隆其MHCIIA基因,初步探究該基因的分子多態(tài)性、組織表達(dá)的特異性以及在胚胎和仔魚(yú)中的差異性表達(dá)模式,以期為齊口裂腹魚(yú)的抗病育種研究提供一定的理論基礎(chǔ)。主要組織相容性復(fù)合體(Major Histocompatibility Complex, MHC)的Ⅱ類分子是脊椎動(dòng)物中一種編碼細(xì)胞表面糖蛋白,具有高度多態(tài)性的基因,且部分基因型與機(jī)體抗病力存在較強(qiáng)的相關(guān)性。本研究首先利用同源克隆和RACE技術(shù)獲得齊口裂腹魚(yú)MHCⅡA基因的cDNA全序列,其次借助測(cè)序法分析了該基因的開(kāi)放閱讀框(ORF區(qū))的多態(tài)性,最后運(yùn)用實(shí)時(shí)熒光定量PCR檢測(cè)其組織分布以及在同一親本的胚胎和仔魚(yú)中表達(dá)情況。主要研究結(jié)果如下：(1)齊口裂腹魚(yú)MHCⅡA基因的cDNA序列全長(zhǎng)為909bp (Genebank登陸號(hào)：KM670437),其中5'-UTR為64bp, ORF區(qū)為711bp,3’-UTR為135bp,并含有典型聚腺苷酸化信號(hào)(AATAAA)和27個(gè)Poly(A)。編碼蛋白含有236個(gè)氨基酸,其推測(cè)的蛋白質(zhì)分子量為25.94Kda,理論等電點(diǎn)為pI=4.60。其預(yù)測(cè)的二級(jí)結(jié)構(gòu)同其它物種具有典型的MHCⅡA的結(jié)構(gòu)特征,包含1個(gè)信號(hào)肽、2個(gè)胞外結(jié)構(gòu)域(α-1和α-2)、1個(gè)跨膜區(qū)和1個(gè)胞質(zhì)區(qū)。編碼蛋白的氨基酸序列存在4個(gè)保守的半胱氨酸殘基、經(jīng)典的氨基酸框架結(jié)構(gòu)GXXXGXXGXXXG以及豐富的磷酸化位點(diǎn),且與建鯉(Cyprinus carpiovar Jian)同源性最高,相似度為83.03%。(2)從8尾健康成魚(yú)中得到17條不同的MHCⅡA基因的cDNA序列,其中最多1尾獲得4條不同序列。分析表明該基因至少編碼15條不同的氨基酸序列,至少含有2個(gè)基因座。其中1條氨基酸序列與其它14條的同源性為89.6%～94.2%,序列間的差異介于5.8%～10.4%之間。同時(shí)在編碼的氨基酸殘基中共發(fā)現(xiàn)50個(gè)變異位點(diǎn),其中35個(gè)變異位點(diǎn)主要集中于α-1區(qū),占變異位點(diǎn)總數(shù)的70%,表明該基因具有高度的多態(tài)性。(3) MHCⅡA基因在所檢測(cè)成魚(yú)的19個(gè)不同臟器組織中均有分布,但表達(dá)量差異顯著,體現(xiàn)出表達(dá)的組織特異性。中腎、頭腎、中腸、前腸、后腸、脾臟、紅肌和心臟中的表達(dá)量較高,腦、卵巢、肝胰臟、鰭條、白肌中的表達(dá)量次之。該基因在中腎中檢測(cè)到最高的表達(dá)水平,其是血液的15.869倍(P0.05)。血液、眼睛、精巢、皮膚、鰓和鰾的表達(dá)量較低,其中表達(dá)量最低的鰾只占血液的38.5%(P0.05)。分析表明該基因的表達(dá)水平與免疫相關(guān)的器官或組織緊密關(guān)聯(lián)。(4)通過(guò)檢測(cè)28個(gè)時(shí)期的胚胎發(fā)現(xiàn),未受精卵、受精到原腸期的胚胎中均檢測(cè)到MHCⅡA基因表達(dá)。但受精1h到8細(xì)胞期的胚胎表達(dá)量都低于未受精卵,表明該基因在齊口裂腹魚(yú)早期胚胎中的表達(dá)水平主要受到母源因子mRNA控制。并且該基因在16細(xì)胞期胚胎中表達(dá)量達(dá)到峰值,是未受精卵的2.259倍(P0.05),表明其表達(dá)水平主要受到合子基因的控制。而胚胎進(jìn)入囊胚期后,該基因表達(dá)量顯著性的降低,僅為未受精卵的8.6%(P0.05),尤其是器官形成階段的胚胎中幾乎檢測(cè)不到該基因的表達(dá),表明此發(fā)育階段的胚胎自身合成的MHCⅡA基因顯著性減少或沒(méi)有表達(dá),可能是MHCⅡB基因表達(dá)量增加所致。(5)通過(guò)檢測(cè)9個(gè)時(shí)期的仔魚(yú)發(fā)現(xiàn),MHCⅡA基因在不同時(shí)期的仔魚(yú)中表達(dá)水平差異顯著。在剛出膜仔魚(yú)中的表達(dá)量只有未受精卵的2%(P0.05)。但在進(jìn)入眼球色素出現(xiàn)期和循環(huán)期的仔魚(yú)中,該基因的表達(dá)量顯著性的增加,分別是未受精卵的7.296和24.794倍(P0.05)。并且在消化道貫通到卵黃囊消失期的仔魚(yú)中,該基因的表達(dá)量顯著性的高于之前時(shí)期,并在背鰭形成期達(dá)到峰值,是未受精卵的968.412倍(P0.05)。這種差異性的表達(dá)模式可能與仔魚(yú)免疫器官或組織的逐漸出現(xiàn)和完善,以及從內(nèi)源性營(yíng)養(yǎng)轉(zhuǎn)換到外源性營(yíng)養(yǎng)有關(guān)。
[Abstract]:In this paper, the MHCIIA gene was cloned, and the polymorphism of the gene, the specificity of the tissue expression and the differential expression pattern in the embryo and larva were preliminarily explored in order to provide a theoretical basis for the study of the disease resistance breeding of the homogenous fishes. The main histocompatibility complex (Major Histocompat) The class II molecule of ibility Complex, MHC is a kind of gene that encodes the surface glycoproteins on the surface of vertebrates, and has a highly polymorphic gene, and there is a strong correlation with the disease resistance of the body. First, the whole sequence of cDNA of the MHC II A gene of the homogenous fishes was obtained by homologous cloning and RACE technology. Secondly, the sequencing method was used. The polymorphism of the open reading frame (ORF region) of the gene was analyzed. The tissue distribution and expression in the same parent embryos and larvae were detected by real time fluorescence quantitative PCR. The main results were as follows: (1) the cDNA sequence of the MHC II A gene of the Homo fishes was 909bp (Genebank landing number: KM670437), and 5'-UTR was 6. 4bp, ORF region is 711bp, 3 '-UTR is 135bp, and contains typical polyadenosine acidification signal (AATAAA) and 27 Poly (A). The encoded protein contains 236 amino acids, its speculating protein molecular weight is 25.94Kda, the theoretical isoelectric point is pI=4.60. its two grade structure and other species have typical MHC II A structure features, including 1 signal peptides, 2 The extracellular domain (alpha -1 and alpha -2), 1 transmembrane regions and 1 cytoplasm regions. The amino acid sequence of the encoded protein contains 4 conserved cysteine residues, the classic amino acid framework GXXXGXXGXXXG and rich phosphorylation sites, and the highest homology with the Cyprinus carpiovar Jian, and the similarity of 83.03%. (2) from 8 tail healthy fish. The cDNA sequences of 17 different MHC II A genes were obtained, of which 4 different sequences were obtained at the maximum of 1 tails. The analysis showed that at least 15 different amino acid sequences were encoded, and at least 2 loci were contained. The homology of the 1 amino acid sequences and the other 14 were 89.6% to 94.2%, and the difference between the sequence sequences was between 5.8% and 10.4%. 50 variation sites were found in the encoded amino acid residues, of which 35 heterotopic points were mainly concentrated in the alpha -1 region, accounting for 70% of the total number of heterotopic points, indicating that the gene was highly polymorphic. (3) the MHC II A gene was distributed in 19 different organs of the detected fish, but the difference of expression was significant, showing the expression of the special tissue. The expression of the middle kidney, the head kidney, the midgut, the midgut, the foregut, the posterior intestine, the spleen, the red muscle and the heart is higher, the expression of the brain, the ovary, the liver, pancreas, the fin, and the white muscle is the highest. The gene is the highest expression in the kidney, which is 15.869 times of the blood (P0.05). The expression of the blood, the eyes, the spermary, the skin, the gills and the swim bladder is low. The lowest swim bladder accounted for only 38.5% (P0.05) of the blood. Analysis showed that the expression level of the gene was closely related to the immune related organs or tissues. (4) the expression of MHC II A gene was detected in unfertilized embryos, unfertilized eggs and embryos fertilized to the primary intestinal stage by detection of 28 embryos. But the expression of embryos fertilized from 1H to 8 cell stage was lower than that of unfertilized. The expression level of the gene in the early embryos of the homozygote fishes was mainly controlled by the mother source factor mRNA, and the expression of the gene reached the peak value in the 16 cell stage embryo, 2.259 times as much as the unfertilized egg (P0.05), indicating that the expression level of the gene was mainly controlled by the zygote gene. The expression of the gene after the embryo entered the blastocyst period. Significantly reduced, only 8.6% (P0.05) of unfertilized eggs, especially in the organogenesis stage, could hardly detect the expression of the gene, indicating that the MHC II A gene of the embryo of this developmental stage was significantly reduced or not expressed, probably due to the increase in the amount of MHC II B gene. (5) by detecting the hair of the larvae of 9 periods. At present, the expression level of MHC II A gene was significantly different in the larvae of different periods. The amount of expression in the newborn larvae was only 2% (P0.05) of unfertilized eggs, but the expression of the gene was significantly increased in the larvae of the eye pigments appearing and circulatory, respectively, 7.296 and 24.794 times (P0.05) of unfertilized eggs, respectively, and in the digestive tract. In the larvae of the yolk sac, the expression of this gene was significantly higher than that of the previous period, and the peak of the dorsal fin formation period was 968.412 times that of the unfertilized egg (P0.05). This differential expression pattern may be developed and perfected gradually with the immune organs or tissues of the larvae, as well as from endogenous nutrition to exogenous camp. It is related to breeding.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S917.4

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