齐口裂腹鱼MHCⅡA基因的克

发布时间：2018-06-03 23:43

本文选题：齐口裂腹鱼 + MHCⅡA　；参考：《四川农业大学》2015年硕士论文

【摘要】：本文以齐口裂腹鱼为研究对象,克隆其MHCIIA基因,初步探究该基因的分子多态性、组织表达的特异性以及在胚胎和仔鱼中的差异性表达模式,以期为齐口裂腹鱼的抗病育种研究提供一定的理论基础。主要组织相容性复合体(Major Histocompatibility Complex, MHC)的Ⅱ类分子是脊椎动物中一种编码细胞表面糖蛋白,具有高度多态性的基因,且部分基因型与机体抗病力存在较强的相关性。本研究首先利用同源克隆和RACE技术获得齐口裂腹鱼MHCⅡA基因的cDNA全序列,其次借助测序法分析了该基因的开放阅读框(ORF区)的多态性,最后运用实时荧光定量PCR检测其组织分布以及在同一亲本的胚胎和仔鱼中表达情况。主要研究结果如下：(1)齐口裂腹鱼MHCⅡA基因的cDNA序列全长为909bp (Genebank登陆号：KM670437),其中5'-UTR为64bp, ORF区为711bp,3’-UTR为135bp,并含有典型聚腺苷酸化信号(AATAAA)和27个Poly(A)。编码蛋白含有236个氨基酸,其推测的蛋白质分子量为25.94Kda,理论等电点为pI=4.60。其预测的二级结构同其它物种具有典型的MHCⅡA的结构特征,包含1个信号肽、2个胞外结构域(α-1和α-2)、1个跨膜区和1个胞质区。编码蛋白的氨基酸序列存在4个保守的半胱氨酸残基、经典的氨基酸框架结构GXXXGXXGXXXG以及丰富的磷酸化位点,且与建鲤(Cyprinus carpiovar Jian)同源性最高,相似度为83.03%。(2)从8尾健康成鱼中得到17条不同的MHCⅡA基因的cDNA序列,其中最多1尾获得4条不同序列。分析表明该基因至少编码15条不同的氨基酸序列,至少含有2个基因座。其中1条氨基酸序列与其它14条的同源性为89.6%～94.2%,序列间的差异介于5.8%～10.4%之间。同时在编码的氨基酸残基中共发现50个变异位点,其中35个变异位点主要集中于α-1区,占变异位点总数的70%,表明该基因具有高度的多态性。(3) MHCⅡA基因在所检测成鱼的19个不同脏器组织中均有分布,但表达量差异显著,体现出表达的组织特异性。中肾、头肾、中肠、前肠、后肠、脾脏、红肌和心脏中的表达量较高,脑、卵巢、肝胰脏、鳍条、白肌中的表达量次之。该基因在中肾中检测到最高的表达水平,其是血液的15.869倍(P0.05)。血液、眼睛、精巢、皮肤、鳃和鳔的表达量较低,其中表达量最低的鳔只占血液的38.5%(P0.05)。分析表明该基因的表达水平与免疫相关的器官或组织紧密关联。(4)通过检测28个时期的胚胎发现,未受精卵、受精到原肠期的胚胎中均检测到MHCⅡA基因表达。但受精1h到8细胞期的胚胎表达量都低于未受精卵,表明该基因在齐口裂腹鱼早期胚胎中的表达水平主要受到母源因子mRNA控制。并且该基因在16细胞期胚胎中表达量达到峰值,是未受精卵的2.259倍(P0.05),表明其表达水平主要受到合子基因的控制。而胚胎进入囊胚期后,该基因表达量显著性的降低,仅为未受精卵的8.6%(P0.05),尤其是器官形成阶段的胚胎中几乎检测不到该基因的表达,表明此发育阶段的胚胎自身合成的MHCⅡA基因显著性减少或没有表达,可能是MHCⅡB基因表达量增加所致。(5)通过检测9个时期的仔鱼发现,MHCⅡA基因在不同时期的仔鱼中表达水平差异显著。在刚出膜仔鱼中的表达量只有未受精卵的2%(P0.05)。但在进入眼球色素出现期和循环期的仔鱼中,该基因的表达量显著性的增加,分别是未受精卵的7.296和24.794倍(P0.05)。并且在消化道贯通到卵黄囊消失期的仔鱼中,该基因的表达量显著性的高于之前时期,并在背鳍形成期达到峰值,是未受精卵的968.412倍(P0.05)。这种差异性的表达模式可能与仔鱼免疫器官或组织的逐渐出现和完善,以及从内源性营养转换到外源性营养有关。
[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.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S917.4

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