【作者】 王师；

【导师】 包振民； 胡景杰；

【作者基本信息】 中国海洋大学 ， 遗传学， 2007， 博士

【摘要】 1栉孔扇贝AFLP遗传连锁图谱的构建应用AFLP技术,本研究首次构建了栉孔扇贝的遗传连锁图谱。共21对引物组合用于AFLP分析。F1子代群体的多态位点比率为62.94%。在检测的783个多态性标记中,317个标记呈1:1分离,190个标记呈3:1分离,其余276个标记呈显著偏分离。应用软件MAPMSKER/EXP进行连锁分析,其中LOD设为2.5且θ设为0.30。雄性图谱含19个连锁群,共94个标记,总长1511.4 cM,覆盖率为66.56%;雌性图谱含20个连锁群,共97个标记,总长1610.2 cM,覆盖率为66.05%。雌雄图谱中AFLP标记的分布相对比较均匀。2栉孔扇贝rDNA家族在自然群体内的动态协同进化模式rDNA家族,作为串联重复多基因家族的一员,各重复单元间呈协同进化。本研究主要通过调查ITS序列的indel(insertion/deletion,即插入/缺失)多态性来研究栉孔扇贝rDNA家族在自然群体内的协同进化过程。在优化的条件下,由indel导致的异源双链分子可以在DHPLC(denaturing high-performance liquid chromatography)分析中被有效鉴别。应用该项技术,我们筛查了栉孔扇贝自然群体40个个体ITS序列的indel多态性。令人惊讶的是,仅7.5%的个体的ITS的组成是纯合的,而其它的个体(92.5%)为杂合的。基于DHPLC分析中不同的峰型,7个个体被选择通过测序的方法来调查个体内的indel多态性。进一步,单精子内indel多态性在更多的不同峰型个体中被调查。基于这些结果,本研究提出一个符合栉孔扇贝rDNA家族在自然群体内协同进化的新模式。不同于以往提出的模式,占群体最高比例的双峰个体可以被看作处于稳定和平衡的状态。这一状态由快速的染色体内重组机制所维持。单峰个体可以被看作处于取得完全均质化状态。它们在群体内仅占很低比例,因而染色体间重组的速度慢于染色体内重组的速度。三峰个体可以被看作处于正均质化一个突变体。该突变体可通过突变或染色体间重组产生。它们在群体内占相当高的比例,这一点或许暗示染色体间重组的速度并不低。3栉海杂交扇贝( Chlamys farreri♀×Argopecten irradians♂)早期发育过程中rDNA家族由偏向性基因转变介导的快速协同进化本研究还调查了栉海杂交扇贝早期发育过程中其基因组内亲本ITS序列的变化。获得的主要结果为:(1)PCR-RFLP分析结果显示杂种基因组内父本ITS的数量随着杂种的发育而逐渐减少,甚至在受精后14天时几乎完全消失;(2)FISH(fluorescence in situ hybridization)分析结果显示在杂种基因组内母本ITS序列被发现存在于父本来源的染色体上,而父本ITS序列却未被发现存在于母本来源的染色体上;(3)应用PCR-RFLP技术在杂种基因组内鉴别出6个重组突变体。由于重组区域比较短,因而位点特异性重组或许参与这一偏向性的基因转变过程。基于这些结果,我们推断rDNA家族在栉海杂种早期发育过程中发生了由偏向性基因转变引发的快速的协同进化。4栉孔扇贝与扇贝科其它7种扇贝ITS1二级结构的系统进化与结构进化分析目前,在序列比对过程中对缺口进行精确定位十分困难。当比对的序列具高度变异性时,这一缺点尤为突出。为了克服这一困难,本研究提出了一个新的策略用于解决基于高变序列的二级结构进行系统推断问题。在这个新策略中,完整的二级结构被直接用作聚类的字符,因而不再需要将其分解成同源的亚结构。本研究的结果显示,可靠的系统发生关系可以从完整的二级结构比对中推断得出,但不能从序列比对中推断得出(即便辅以结构的信息)。因而本研究结果充分支持这一新策略的在实践上的可行性。此外,本研究还调查了扇贝科8种扇贝ITS1二级结构的进化情况。完整的ITS1二级结构可以被分为4个结构域。互补性的碱基变化被发现存在于这4个结构域中。进一步,4个结构域内保守的结构模序被鉴别出来。这些模序,特别是D2和D3中的模序,或许在rRNA的成熟过程中起着重要的作用。5栉孔扇贝和虾夷扇贝Ty3/Gypsy类群Mag系反转座子CFG1和PYG1的克隆与特性分析本研究首次从栉孔扇贝和虾夷扇贝基因组内克隆得到Ty3/Gypsy类群Mag系的两个反转座子CFG1和PYG1。CFG1元件的全长为4826bp,包括5’-LTR(192bp),完整的ORF(4047bp)和3’-LTR(189bp)。CFG1和PYG1元件完整的ORF包含1348个氨基酸,未发现阅读框移位现象。与它们关系最近的是花斑剑尾鱼的Jule元件。栉孔扇贝二倍体基因组约含84个拷贝的CFG1元件。对于CFG1,PYG1和Ty3/Gypsy类群Mag系的其它元件,本研究总结了它们的主要特征,并比较了它们反转录酶(RT)域的3D结构。CFG1元件在幼虫期mRNA的表达量的变化规律为:在原肠胚前,其逐渐升高;在原肠胚后,其逐渐降低。而CFG1元件在闭壳肌和消化腺组织中mRNA的表达量比成体的其它组织略微低一些。总的来看,CFG1元件mRNA的表达量在幼虫期显著高于其在成体组织。CFG1元件的启动子和部分组特异性抗原(GAG)域处于未甲基化状态,而部分RT域则处于高度甲基化状态。因而,CFG1的表达可能是由转录后抑制机制而不是甲基化所控制。更多还原

【Abstract】 1. Construction of AFLP linkage map for C. farreriThe linkage maps of C. farreri were constructed with AFLP markers generated from 21 primer combinations. The polymorphism level of F1 progeny is 62.94%. Of the 783 polymorphic markers detected, 317 segregated in 1:1 ratios and 190 in 3:1 ratios, whereas the other 276 showed segregation distortion. Linkage analysis used the program MAPMAKER/EXP at LOD=2.5 andθ=0.30. The male map containing 94 markers in 19 linkage groups, was 1511.4 cM in total length and 66.56% in genome coverage. The female map containing 97 markers in 20 linkage groups, was 1610.2 cM in total length and 66.05% in genome coverage. The distribution of AFLP markers is relatively even in chromosomes of male and female map.2. Dynamics of concerted evolution of rDNA family in natural population of C. farrerirDNA family, as one of tandemly repeated families, exhibits the general pattern of concerted evolution. In order to study the homogenization process of rDNA family in the population of Zhikong scallop, this study focused on the insertion/deletion (indel) polymorphism of the internal transcribed spacer (ITS) sequence. Under the optimized condition, ITS heteroduplexes caused by indels could be well identified in denaturing high-performance liquid chromatography (DHPLC) analysis. Using this technique, ITS indel polymorphism in 40 individuals from natural population was investigated. Surprisingly, only 7.5 percent of individuals were homogeneous in ITS constitution, while the others (92.5%) were heterozygous. Based on peak types appeared in DHPLC analysis, seven representative individuals were chosen and ITS polymorphism within an individual was then investigated using sequencing method. Further, ITS indel polymorphism within single sperm was investigated in more individuals. Based on these results, a new model was proposed for concerted evolution of rDNA family in natural population of C. farreri. Different from previous models, 2-peak individuals with the highest proportion (62.5%) in natural population can be seen as being in a steady and balanced state maintained by rapid intrachromosomal recombination. 1-peak individuals can be seen as being in the state of full homogenization. They occupy the low proportion (7.5%) in natural population, which suggests that interchromosomal recombination is slower than intrachromosomal recombination. 3-peak individuals can be seen as being in the state of homogenizing a variant which may be generated by mutation or interchromosomal recombination. They occupy relatively high proportion in natural population, which implies that the rate of interchromosomal recombination may be not rare.3. Rapid concerted evolution via biased gene conversion occurs in the rDNA family of a hybrid scallop (Chlamys farreri♀×Argopecten irradians♂) at the early developmental stageThis study also focused on the variation of the parental ITSs in a hybrid scallop (Chlamys farreri♀×Argopecten irradians♂) at the early developmental stage. The main results obtained in this study are: (i) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that the quantity of paternal ITS in the hybrid had decreased gradually accompanying the development of the hybrid and even nearly disappeared at the 14th day after fertilization; (ii) fluorescence in situ hybridization (FISH) analysis in the hybrid revealed that maternal ITS had been found in the paternal ITS-bearing chromosomes, while paternal ITS had not been found in the maternal ITS-bearing chromosome; (iii) with PCR-RFLP technique, six recombinant variants were found in the hybrid and identification of short recombination regions implied that site-specific recombination may be involved in the biased gene conversion. Based on these results, it was concluded that rapid concerted evolution via biased gene conversion had occurred in the rDNA family of the hybrid at the early developmental stage.4. Analysis of the secondary structure of ITS1 in Pectinidae: implications for phylogenetic reconstruction and structural evolutionIt is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, this study developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into 4 structural domains. Compensatory changes were found in all 4 structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.5. Cloning and characterization of two novel elements (CFG1 and PYG1) of Mag lineage of Ty3/Gypsy retrotransposons from Zhikong scallop (Chlamys farreri) and Japanese scallop (Patinopecten yessoensis)Two novel elements (CFG1 and PYG1) which belong to Mag lineage of Ty3/Gypsy retrotransposons were cloned from Zhikong scallop (Chlamys farreri) and Japanese scallop (Patinopecten yessoensis). The total length of CFG1 element is 4826 bp, including 5’-LTR (192 bp), entire ORF (4047 bp) and 3’-LTR (189 bp). The entire ORFs of both CFG1 and PYG1 elements are composed of 1348 aa and do not have any frameshifts. Their closest relative is Jule element from the poeciliid fish (Xiphophorus maculatus). On average, the diploid genome of C. farreri contains approximate 84 copies of CFG1 elements. For CFG1, PYG1 and other elements of Mag lineage of Ty3/Gypsy group, this study summarized their major features and compared their 3D structures of RT domains. The mRNA expression of CFG1 element in larvae increases gradually before gastrulae stage and decreases gradually after gastrulae stage, whereas expressions in adductor muscle and digestive gland are relatively lower than those in other tissues of adults. Overall, the mRNA expression of CFG1 element in the early larvae is significantly higher than those in adult tissues. The promoter and partial GAG domain of CFG1 element are unmethylated, however, the partial RT domain is highly methylated. Therefore, it seems that CFG1 expression may be controlled by post-transcriptional suppression rather than methylation.更多还原