【作者】 李春梅；

【导师】 张全启；

【作者基本信息】 中国海洋大学 ， 遗传学， 2008， 硕士

【摘要】 半滑舌鳎(Cynoglossus semilaevis)是一种重要的经济鱼类,具有生长快等优点。近年来,对其研究主要集中在养殖、发育、群体遗传等方面,对其重要的功能基因如免疫相关基因还知之甚少。主要组织相容性复合体(MHC)是脊椎动物中普遍存在的编码免疫球蛋白样受体的高度多态的基因群,因其在免疫系统中的重要作用而备受关注。目前已在多种鱼类中扩增得到MHC基因。本文利用RACE技术得到半滑舌鳎MHC II类B基因的cDNA全长,同时扩增研究了其多态性、发现了该基因的两型、获得了两型的基因全长和表达情况等。首先,从实验室构建的半滑舌鳎cDNA文库中得到了MHC II类B基因的完整开放阅读框(Open Reading Frame,ORF),利用RACE(Rapid amplification of cDNA ends,RACE)技术得到了该基因的cDNA全长,共1215bp,其中ORF 750bp,5’末端非翻译区(Untranslated region,UTR)28bp,3’UTR 437bp。在编码的249个氨基酸中,前21个氨基酸组成信号肽,第215到237位氨基酸为跨膜区域,在β1和β2区有4个保守的半胱氨酸残基。生物软件分析表明,该基因编码的氨基酸有1个N连接的糖基化位点、4个蛋白激酶C磷酸化位点、6个酪蛋白激酶II磷酸化位点、2个酪氨酸激酶磷酸化位点、4个豆蔻酰化位点等保守结构;二级结构显示该基因编码的蛋白为混合型蛋白;三维结构和人的DRB的三维结构非常相似。序列比对发现半滑舌鳎MHC II B蛋白和牙鲆、大菱鲆、三刺棘鱼等的MHC II B一致性较高;进化树中首先跟三刺棘鱼聚在一起,然后和牙鲆、大菱鲆、大黄鱼等相聚。设计位于ORF两侧的引物,用高保真酶从9条鱼中扩增该基因的ORF,挑取阳性克隆进行双向测序,结果得到24个非重复序列;这些序列在核苷酸水平上存在明显差异,进化树中聚为两支,分别命名为MhcCyse-DAB1和MhcCyse-DAB2 ,其中MhcCyse-DAB1包含14种等位基因(Cyse-DAB1*0101-Cyse-DAB1*1401),MhcCyse-DAB2包含8种等位基因(Cyse-DAB2*0101- Cyse-DAB2*0801);两型均在β1区的变异最大,且核苷酸变异多为非同义替换,因此,24种不同的核苷酸序列产生了22种等位基因;还有两个序列在氨基酸水平上分别和Cyse-DAB1*0101、Cyse-DAB2*0201一样,但核苷酸序列存在差异,将其分别命名为Cyse-DAB1*0102、Cyse-DAB2*0202。其中Cyse-DAB1各等位基因的距离为0.4-6.7,Cyse-DAB2各等位基因的距离为0.4-8.5;Cyse-DAB1各等位基因与Cyse-DAB2各等位基因的距离为8.5-16.6。另外,还发现两个缺失突变的等位基因,分别缺失2bp和7bp,推测为假基因,在核酸水平上分别属于Cyse-DAB1和Cyse-DAB2,因此分别命名为Cyse-DAB1-pse1、Cyse-DAB2-pse1。在检测的9条鱼的mRNA水平上除1条检测不到Cyse-DAB2的表达外,其余均能检测到两型的表达。同时,克隆研究了其中一个个体的3’UTR,发现有2种3’UTR序列,Cyse-DAB1和Cyse-DAB2各有1种,表明两型各至少存在一个位点;但两型之间的3’UTR区差异不大,仅存在2个核苷酸变异和1bp长度差异。随后扩增研究了5个个体的内含子1,得到两型的内含子1各5种,每型的内含子1分别聚成一支,差异明显;表明两型各至少有5个位点,个体分析表明每个个体每型各至少有1-2个位点。利用5’和3’UTR的引物和Extaq扩增得到两型的基因全长。两型均由6个外显子、5个内含子组成,均约2.4kb;在外显子的长度和分布上完全一致,但两型之间和型内内含子的长度和核苷酸存在较明显差异。两型的内含子除内含子1可以作为划分两型的依据外,其它内含子因差异不明显或存在两型的交叉,都不适合作为划分依据。在内含子1、2、4中发现了微卫星序列。内含子的多态也再次说明了该基因的高度多态现象。在mRNA水平检测不到Cyse-DAB2表达的S1个体,也扩增得到了Cyse-DAB2基因全长;没有发现剪切突变或缺失\插入突变,推测Cyse-DAB2的表达可能和生理状态有关。用RT-PCR分别检测两型在不同组织和不同发育时期的表达,结果发现两型在不同组织的表达情况类似,在大多数组织中(肝、脾、肾、鳃、脑、心、肠、卵巢、精巢)都表达,而且在脾、肾和心中表达量都较高,在肌肉和鳍中检测不到;但是Cyse-DAB2在鳃、肠、卵巢和精巢的表达不及Cyse-DAB1高。在发育时期中,Cyse-DAB1在受精卵、原肠胚、眼芽和孵化后5-22天有表达,且在孵化后5天表达开始呈现略微下降的趋势;但相同条件下检测不到Cyse-DAB2型在发育时期的表达,说明两型在功能上存在一些差异。更多还原

【Abstract】 Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish, and has many strongpoints such as grow fast in aquaculture. Recently, there are many studies focus on its aquaculture, development and population genetics; but little is known about its important genes such as immune-related genes. Major histocompatibility complex(MHC)encodes immunoglobulin-like receptors in nearly all vertebrate and is highly polymorphic gene family. It is given great attention for its important roles in immune systems. At present, many MHC genes are studied in several kinds of fishes. This paper discussed the cDNA of MHC class II B got from half-smooth tongue sole, studied its polymorphism, discovered the two subtypes, and studied the two subtypes’specific genomic organization and expression pattern.First of all, the Open Reading Frame (ORF) of MHC class II B was got from the cDNA library of half-smooth tongue sole constructed by the lab, and then the full cDNA sequence was got by using RACE (Rapid amplification of cDNA ends, RACE). The cDNA comprises a 28bp 5’UTR (Untranslated region, UTR), a 750bp ORF and a 437bp 3’UTR. The first 21 putative amino acids form the signal peptide, and the 215th-237th amino acids constitute the transmembrane region. There are four conserved cysteines in theβ1 andβ2 domain; and also one N-glycosylation site, four protein kinase C phosphorylation site, six casein kinase II phosphorylation site, two tyrosine kinase phosphorylation site and four N-myristoylation site in the putative amino acid. The secondary structure shows that class II B protein is a mixed protein, and its tertiary structure is very similar to HLA-DRB’. Its putative amino acid identity is quite high with the same gene from three-spined stickleback, Japanese flounder and turbot; and it clusters first with that from three-spined stickleback, then with Japanese flounder, turbot and large yellow croaker in phylogenetic tree, which demonstrate half-smooth tongue sole’s closer relationship with those fishes.Then ORF polymorphism was studied by using primers in 5’UTR and 3’UTR and high fidelity polymerase from 9 individuals. Positive clones were sequenced from both directions, which resulted in 24 nonredundant sequences. There are apparent divergence among those sequences, which cluster into two subgroups, and named as MhcCyse-DAB1 and MhcCyse-DAB2, respectively. MhcCyse-DAB1 includes 14 alleles, while MhcCyse-DAB2 includes 8 alleles; and the most variable region isβ1 domain. Most of the substitutions among alleles are nonsynonymous, which lead to 22 alleles from the 24 sequences. There are also two sequences, which differ from all the other sequences in nucleotides, but have the same putative amino acids as Cyse-DAB1*0101 and Cyse-DAB2*0201, respectively, so they are named as Cyse-DAB1*0102 and Cyse-DAB2*0202, respectively. The intragroup alleles’divergence in Cyse-DAB1 and Cyse-DAB2 is 0.4-6.7 and 0.4-8.5, respectively; and the intergroup alleles between Cyse-DAB1 and Cyse-DAB2 is 8.5-16.6. Still, there are another two sequences which has a deletion mutation of 2bp and 7bp, respectively; and are presumed as pseudogenes. For they have similar nucleotide sequence as from Cyse-DAB1 and Cyse-DAB2, so they are named as Cyse-DAB1-pse1 and Cyse-DAB2-pse1, respectively. And most of the individuals expresse both of the two subtypes, except the S1 individual which cannot be detected the Cyse-DAB2’s expression..At the same time, 3’UTR was studied and two kinds of 3’UTR sequences were discovered. It turns out that each subgroup own one 3’UTR sequence, which indicates that each subgroup has at least one locus. However, the divergence between the two 3’UTR sequence is small, which includes only two basepair variations and 1bp difference in length.The study of intron 1 from 5 individuals indicates similar results: intron 1 from the same subgroup cluster first and distinct difference was discovered between the two subgroups. Five kinds of Cyse-DAB1 intron1 and five kinds of Cyse-DAB2 intron1 sequences were got, which indicate that each subgroup has at least five loci and individual study shows that each individual has at least one to two loci of each subgroup.The genomic whole length was got by primers in 5’UTR and 3’UTR and Extaq. It turns out that both the two subtypes consist of six exons and five introns, both of which span about 2.4kb. The two subtypes have the same exon length and distribution, but have great divergence in the intron length and sequence. It shows that only intron 1 can be the parameter to distinguish the subgroups, all others cannot for the similarity and crossover in the nucleotide sequences. There are also microsatellites in intron 1, 2 and 4. The genomic sequence of Cyse-DAB2 from S1 individual was also got, and no mutation in RNA splicing and no deletion\insertion mutation in ORF was discovered, which show that the expression of Cyse-DAB2 may be relevant to the body’s physiological environment.The expression profile of different tissues and developmental stages were studied by using RT-PCR. The two subtypes have similar expression pattern in different tissues, and express in most tissues (liver, spleen, kidney, gill, brain, heart, intestine, ovary and testis), and have high expression in spleen, kidney and heart; while the expression in muscle and fin cannot be detected and Cyse-DAB2 has a lower expression in gill, intestine, ovary and testis than Cyse-DAB1. In developmental stages, Cyse-DAB1 can be detected in fertilized egg, gastrula stage, eye bud stage and 5-22 day after hatching, and has a drop from 5 day after hatching; while Cyse-DAB2 cannot be detected under the same condition, which indicate that the two subtypes may have different functions.更多还原