节点文献
半滑舌鳎MHC Ⅱ类基因的克隆、与抗病相关性及遗传连锁分析
【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， 2010， 博士
【摘要】 半滑舌鳎(Cynoglossus semilaevis)是一种重要的经济鱼类,在我国沿海的鱼类养殖中占有重要地位。养殖中的半滑舌鳎会面临很多病原的侵袭。因此,为了解其重要免疫基因,从根本上提高鱼体自身的抗病能力,我们克隆了主要组织相容性复合体(MHC)的Ⅱ类基因,并研究了A基因的开放阅读框(open reading frame, ORF)多态、基因结构及B基因在不同发育时期的表达；对200尾个体进行了鳗弧菌注射实验,以期找到与抗鳗弧菌感染相关的等位基因；同时利用3个家系研究了Ⅱ类基因的不同等位基因之间的连锁关系。主要结果如下：1.Ⅱ类的两个B基因在不同发育时期的表达之前的研究结果表明,在半滑舌鳎中B基因有两个,即Cyse-DAB和Cyse-DBB。用这两个B基因的特异引物分别对半滑舌鳎的19个发育时期进行扩增,结果发现两个B基因的表达不同。相同条件下,Cyse-DAB在受精后至胚环期未检测到表达,从尾牙期表达开始上升并持续到孵化后2d,而后在孵化后5d表达下降,在16d未检测到；在未变态时期表达量很高；Cyse-DBB在从未受精卵开始一直到孵化后2d均有持续表达,到孵化后5d表达未检测到,在孵化后16d表达量很大；两个B基因都随着变态的进行表达量下降,处于正在变态时期的表达量较小,而后随着变态的结束,表达量逐渐恢复,在56d和87d仔鱼时期表达量均较大。两个B基因在发育早期的不同表达情况说明在这些时期两个B基因可能发挥不同的功能或功能互补。2.Ⅱ类A基因的克隆、ORF多态和基因结构利用同源克隆技术,从半滑舌鳎的脾脏中克隆得到了A基因的cDNA全长,为992bp,ORF长717bp,编码238个氨基酸。一级结构显示其有免疫球蛋白和主要组织相容性复合体蛋白标签和保守的半胱氨酸残基；二级结构显示其为混合型蛋白。序列比对和进化树分析显示,半滑舌鳎的该蛋白和大菱鲆、牙鲆、大西洋庸鲽的亲缘关系最近。对9个个体进行了A基因的ORF扩增,得到了25种多肽序列,序列比对和进化树分析显示这些多肽序列差异明显,分为两类,应为两个基因,将其分别命名为Cyse-DAA和Cyse-DBA,分别包含11个和14个等位基因；两个基因的特异氨基酸分别为Asn91、Ser92、Ser94、Gly95、Lys101、Lys103和Thr/Ala92、Glu/Gln94、Ala95、Leu101、Asn103。与其它有代表性的含有2个位点以上的鱼类A基因构建进化树发现,半滑舌鳎的这两个A基因的各等位基因分别聚在一起,形成两个分支；随后与其它物种的A基因聚在一起；提示这两个A基因是在半滑舌鳎与其它物种分离后才产生的。对两个A基因的基因全长扩增显示,两个A基因均由4个外显子和3个内含子组成,外显子的分布和长度一致,但内含子差异明显：Cyse-DAA的内含子2长为136bp,而Cyse-DBA的内含子2长为205bp,因而内含子2可以用来区分两个A基因；两个A基因的内含子1和3没有明显差别,且内含子3最为保守均为90bp；内含子1含有一个重复次数约为100次的(GTCA)微卫星序列。3.感染实验和抗病/易感等位基因的查找对200尾半滑舌鳎进行腹腔注射鳗弧菌实验,取最先死亡的20尾个体和活跃的20尾个体进行分析。每个个体每个基因各挑取5个克隆进行测序。共发现了B基因的多肽序列60个,其中属于Cyse-DAB的为32个,属于Cyse-DAB的为28个；两个B基因有各自特异的核苷酸和氨基酸；发现了13种CCyse-DAB的内含子1和11种Cyse-DAB的内含子1；Cyse-DAB的等位基因Cyse-DAB*0701和Cyse-DAB-1301与抗鳗弧菌感染的敏感性相关,且差异显著,P分别为0.001和0.01；虽然Cyse-DAB的等位基因Cyse-DAB*0601、Cyse-DAB*0706、Cyse-DBB的等位基因Cyse-DBB*0101和Cyse-DBB*1301与抗鳗弧菌感染的抗性相关性不显著,但是这四个等位基因很可能与抗鳗弧菌感染的抗性相关,因为它们仅在存活个体中被发现,且在多于一个个体中被发现。根据得到的外显子2、3的的氨基酸序列共发现了85种A基因的多肽序列,其中的41种属于Cyse-DAA,45种属于Cyse-DBA；两个A基因在特异位置的特异氨基酸保守；两个A基因的内含子2长度保守,本实验中得到了3种CCyse-DAA的内含子2和6种Cyse-DBA的内含子2,差异主要为单核苷酸变异；虽然没有显著性差异,但是Cyse-DAA的等位基因Cyse-DAA*0901、Cyse-DBA的等位基因Cyse-DBA*1101和Cyse-DBA*1401在死亡个体中比在存活个体中出现的频率高,可能与抗鳗弧菌感染的敏感性相关；而Cyse-DAA的等位基因Cyse-DAA*0201、Cyse-DAA*1101、Cyse-DBA等位基因Cyse-DBA*0401、 Cyse-DBA*1102、Cyse-DBA*1801以及Cyse-DBA*2201在存活个体中出现的频率比在死亡个体中出现的频率高,因而可能与抗鳗弧菌感染的抗性有关。4Ⅱ类基因的等位基因的连锁分析虽然用PstI酶切半滑舌鳎基因组后的Southern杂交结果显示B基因只有一条大于lOkb的条带出现,但是序列分析仍然支持半滑舌鳎中存在2个B基因的结论。采用多重PCR的方法对三个家系的子代进行B基因的不同等位基因间引物组合的扩增,结果显示：Cyse-DAB的等位基因Cyse-DAB*0403和Cyse-DAB*0601连锁,重组率8.00%；Cyse-DAB的等位基因Cyse-DAB*0601(?)口Cyse-DBB的等位基因Cyse-DBB*0101紧密连锁；(Cyse-DBB的等位基因Cyse-DBB*0101和Cyse-DBB*1601连锁,重组率4.17%；Cyse-DAB的等位基因Cyse-DAB*2401和Cyse-DBB的等位基因Cyse-DBB*2501紧密连锁；Cyse-DAB的等位基因CCyse-DAB*0403和Cyse-DBB的等位基因Cyse-DBB*0501连锁,重组率14.00%。这些信息表明B的两个基因以Cyse-DAB和CCyse-DBB紧密连锁在一起为一个单元,在染色体上至少串联重复一次。对三个家系的各约4个子代个体进行B基因测序发现了等位基因从亲本到子代的遗传。由于A基因的核苷酸水平的变异位点非常多,不能像B基因分析中用多重PCR的方法,因而设计单个等位基因的引物在子代中扩增,并与同亲本来源的B基因的等位基因进行连锁分析,结果显示2#家系母本的Cyse-DAA的等位基因Cyse-DAA*2201与Cyse-DAB的的等位基因Cyse-DAB*0601、Cyse-DBB的等位基因Cyse-DBB*0101及Cyse-DBB*1601的距离较远,属于自由组合；3#家系父本的Cyse-DBA的等位基因Cyse-DBA*3101与Cyse-DAB的的等位基因Cyse-DAB*0403及Cyse-DBB的等位基因Cyse-DBB*0501的距离较远,属于自由组合。同时发现亲本的A基因的等位基因在子代间得到很好的分离和遗传。
【Abstract】 Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish, and accounts for a high percentage in fish aquaculture in coastal area of China. Half-smooth tongue sole has to face many kinds of pathogens during its growth in aquiculture. Hence, in order to understand its main immune genes and improve its anti-disease ability, we cloned the major histocompatibility complex (MHC) class II genes from half-smooth tongue sole, and studied the open reading frame (ORF) polymorphism, genomic structure of A gene and also the developmental stages expression of B gene; moreover,200half-smooth tongue sole individuals were injected with Vibrio anguillarum in order to find the potential anti-bacterial or susceptible alleles; meanwhile, three families were used to analyze the linkage relationship of different alleles of class II genes. The main results are listed below:1. Expression study of two B genes in developmental stagesAccording to previous study, there are two B genes in half-smooth tongue sole, named as Cyse-DAB and Cyse-DBB. Diffferent expression patterns were found in19developmental stages using the two genes’ specific primes. Cyse-DAB was undetectable from fertilized eggs to middle gastrula stage. Its expression was observed again from tail-bud stage and increased until2-d larvae, then it decreased in5-d larvae and could not be detected in16-d larvae, but the expression of Cyse-DAB was high just before metamorphosis. Cyse-DBB had a continuous expression from unfertilized eggs to2-d larva. The expression of Cyse-DBB was not detected in5-d larvae, but high expression was detected in16-d larvae. The expression of both genes decreased when metamorphosis began, and had low expression during metamorphosis; then the expression increased and high expression was found in56-d and87-d fingerlings. The different expression patterns in early developmental stages indicated that the two B genes might play different or complementary roles in those stages.2. Cloning, ORF polymorphism and genomic structure of class ⅡA geneA992bp cDNA encoding A gene was cloned from spleen of half-smooth tongue sole, and the ORF is717bp in length, encoding a pepetide of238amino acids. The immunoglobulins and major histocompatibility complex proteins signature and conserved cysteines were identified in the primary structure of the A protein. The secondary structure indicates that A protein is a complex protein. Sequence alignment and phylogenetic analysis indicate that half-smooth tongue sole A protein has a close relationship with that from turbot, Japanese flounder and Atlantic halibut.Nine individuals were used to study the ORF polymorphism and25peptides were identified. Sequence alignment and phylogenetic analysis indicate that those peptides had divergent sequences, clustered into two groups, so they should belong to two A genes and named as Cyse-DAA and Cyse-DBA accordingly. Cyse-DAA and Cyse-DBA has11and14alleles, respectively. The two A genes’specific amino acids in specific positions are Asn91, Ser92, Ser94, Gly95, Lys101and Lys103for Cyse-DAA, and Thr/Ala91, Val/Gly92, Glu/Gln94, Ala95, Leu101and Asn103for Cyse-DBA. Phylogenetic analysis with other teleosts who has at least two A loci indicates that the two A genes’alleles from half-smooth tongue sole clustered into two groups, then joined as a big branch, then with other A genes from other species, which suggests the originating time of two A genes should be after half-smooth tongue sole seperating from other species.The whole genomic sequences indicate that both of the two A genes are comprised of four exons and three introns, and the distribution and length of the exons are same, but the introns are quite different:Cyse-DAA’s intron2is136bp in length, while Cyse-DBA’s intron2is205bp long; but the two genes’intron1and intron3are not quite divergent. Intron3is well conserved, all are90bp long; while in intron1a (GTCA) repeat of about100times was discovered.3. Vibrio anguillarum infection and the search for anti-bacterial/susceptible allelesTwo hundreds half-smooth tongue sole individuals were selected for the V. anguillarum intraperitoneal infection experiment. The first20died individuals and20active ones were screened for the class II genes’alleles. Five clones of each gene were sequenced from each individual. Then60peptides of B genes’were identified, including32Cyse-DAB alleles and28Cyse-DBB alleles. The two B genes have specific nucleotides and amino acids in specific positions. And13Cyse-DAB intron1and11Cyse-DBB intron1sequences were found. Cyse-DAB’s allele Cyse-DAB*0701and Cyse-DAB*1301are potential susceptible alleles with significant difference (P value is0.001and0.01, respectively). Though the difference is not significant in Cyse-DAB’s allele Cyse-DAB*0601, Cyse-DAB*0706, Cyse-DBB’s allele Cyse-DBB*0101and Cyse-DBB*1301, these four alleles are potential anti-bacterial alleles for they were only detected in several surviving individuals.According to the amino acids in exon2and exon3,85peptides of A genes’were identified, including41Cyse-DAA alleles and45Cyse-DBA alleles. The two A genes’ specific nucleotides and amino acids are well conserved. The length of intron2of the two A genes are also well conserved, and three Cyse-DAA intron2and six Cyse-DBA intron2sequences were found because of single nucleotide polymorphism. Though the difference is not significant, Cyse-DAA’s allele Cyse-DAA*0901, Cyse-DBA’s allele Cyse-DBA*1101and Cyse-DBA*1401are potential susceptible alleles with higher frequencies in dead individuals, and Cyse-DAA’s allele Cyse-DAA*0201, Cyse-DAA*1101, Cyse-DBA’s allele Cyse-DBA*0401, Cyse-DBA*1102, Cyse-DBA*1801and Cyse-DBA*2201are potential anti-bacterial alleles for they had higher frequencies in surviving individuals than dead ones.4. Linkage analysis of alleles from class II genesThough only one fragment was detected by Southern blot analysis after PstI digestion using B genes’probe, the different sequences of B genes support the conclusion that two B genes exist in half-smooth tongue sole. Three families were used for linkage analysis. Using poly-PCR and different primer combination, the linkage relationships are as follows:Cyse-DAB’s allele Cyse-DAB*0403and Cyse-DAB*0601are linked with a recombination fraction of8.00%; Cyse-DAB’s allele Cyse-DAB*0601and Cyse-DBB’s allele Cyse-DBB*0101are tightly linked; Cyse-DBB’s allele Cyse-DBB*0101and Cyse-DBB*1601are linked with a recombination fraction of4.17%; Cyse-DAB’s allele Cyse-DAB*2401and Cyse-DBB’s allele Cyse-DBB*2501are tightly linked; Cyse-DAB’s allele Cyse-DAB*0403and Cyse-DBB’s allele Cyse-DBB*0501are linked with a recombination fraction of14.00%. These information indicates that the two B genes repeat at least twice in one chromosome with the unit of a tightly linked Cyse-DAB and Cyse-DBB. Sequencing of B genes from about four offsprings from each family indicates allele’s inheritance from parents to the offsprings.For the nucleotide variations in A genes are quite high, so poly-PCR is not suitable here and only one allele’s specific primer could be used in each PCR. Compared with the B genes’alleles from the same parent indicated that Cyse-DAA’s allele Cyse-DAA*2201are not linked with three of B’s sequences (Cyse-DAB*0601, Cyse-DBB*0101and Cyse-DBB*1601) in family2, and Cyse-DBA’s allele Cyse-DBA*3101are not linked with two of B’s sequences (Cyse-DAB*0403and Cyse-DBB*0501) in family3for the high recombination fraction near50%. Sequencing of A genes in the offsprings indicates alleles have good inheritance and separation from parents to the offsprings.