Study On Inheritance Divergence Of Transposons In Polyploidy Fish And Gonad Development Of Gynogenetic Fish In Molecular Biology

Transposons are discrete segments of DNA capable of moving through the genome of their host,which play an important role of species evolution.Transposons can jump from an old site into a new site in the genome of its host and change the structures of gene and genome, providing the materials for the generation of polymorphism and speciation. The study on transposons was significant in the many biological issues, such as tranposon structure and function,the origin and evolution of species,genomic polymorphism,the orderly progression of gene expression regulation,etc.Female red crucian carp Carassius auratus red var matched with male blunt snout bream Megalobrama amblycephala, successful generating triploid and tetraploid hybrids.In the present study, the number of DNA transposons in the genomes of tetraploid hybrids, triploid hybrids and biploid parents of female red crucian carp and male blunt snout bream were detected,and the molecular structure and genetic characteristic of TC1 transposons were illuminated.Nucleotide and amino acid identity comparisons were performed between the sequences of the gene coding a high mobility group protein in fish with different ploidy level.The interaction between this protein and DNA transposons were analyzed.Based on allotetraploid hybrids and gynogenetic method,the gynogenetic clone lines were obtained,and then explored by suppression subtractive hybridization method in order to study the molecular mechanism underlying diploid hybrids producing diploid eggs.The major results in this paper were presented as follows:1.DNA contents of the samples were measured using a flow cytometer.Mean values for the haploid genome sizes(C values) of blunt snout bream are 1.55 pg/nucleus(1438Mb).A novel transposon designated as Tmal was found in the genome of this sample,consisting of 670 copies by Dot-blot analysis,and tested by Southern blot.This element was a member of the third group in Tel family.Compared with the reconstructed and active transposon in salmon fish,the conceptual amino acid sequences of Tmal was shown that it harbored a paired-like domain with Leucine-zipper,a GRRR-like putative AT-hook motif,and bipartite nuclear localization signal motifs.This element was inactivity of transposition in its host genome because of the multiple internal stop codons.This is the first report that DNA transposons were found in the genome of blunt snout bream.2.The crosses of the distantly-related cyprinids species were performed using blunt snout bream as male parent and red crucian carp as female parent,and in so doing to result in the excision of transposons Tmal from the offspring genome of tetraploid hybrids;while Tmal in the triploid hybrid's genome only contained conversion and insertion mutations.These results suggest that the transposon generated two types of genetic variation in this cross-breeding:one is vertical inactivation (with insertion and point mutations responsible for this) and the other is stochastic loss(transposon escaped in going from its parents to progeny).3.A novel tranposon named Ttel was found in the genome of tetraploid hybrids by PCR method.There were 880 copies of this element in the genome of tetraploid hybrids,in which the C values are 1.76 gp / nucleus.This is the first report that the burst of a novel transposon occurred during the intersecific hybridization in vertebrate. 4.Using RACE method,the full-length mRNA of high mobility group protein 1 code gene(HMG1) was obtained from red crucian carp, blunt snout bream and their crossed progeny including triploid and tetraploid hybrids,in which the open reading frame is composed of 579 nucleotides and codes 193 amino acids.Identity comparison of HMG1 indicates that the nucleotide identity(99%) between tetraploid hybrids and its female parent red crucian carp is higher than that(97%) between tetraploid hybrids and its male parent brunt snout bream;the nucleotide identity(95%) between triploid hybrids and its each parent is lower than that(98%) between its female and male parents.In the same way,the protein identity(100%) between tetraploid hybrids and its each parent is higher than that(97%) between triploid hybrids and its each parent.These results are shown that the interspecific hybridization generates a shock to HMG1 in triploid hybrids,causing divergence of nucleotides.The HMG1 protein of tetraploid hybrids was consistent with that of its both parents, helpful for the hybrids to pass by the barrier of the cross incompatibility between alleles,and providing the material basis of the bisexual fertile tetraploid hybrids being sustained from one to another.5.Suppression subtractive hybridization was performed in this study. The cDNAs from the ovary in proliferation phase of the third gynogenesis (G₃) generated by allotetraploids hybrids were employed as the "Driver", and those in growth phase of the fourth gynogenesis(G₄) were used as the "Tester".A forward suppression subtractive hybridization cDNA library was constructed.Seventy-three cDNA clones that are specifically expressed in proliferation phase of G₄ were detected by dot blotting. Sequencing and BLASTX analyses revealed that several of these cDNAs have high homologies to the known sequences in the NCBI database. Their encoded proteins include the signal recognition particle 9,the ATP-binding cassette transporter,the glucanase-xylanase fusion protein, the protein preventing mitosis catastrophe and et al.The up expression profiles of these genes in proliferation phase were confirmed by reverse transcriptase-polymerase chain reaction.Our studies provide insights into the molecular mechanism underlying the ovary development of diploidy hybrid fish of crucian carp×common carp.6.The expression profiles of the gene coding the protein preventing mitosis catastrophe(PMC) at different time-points were analyzed by Quantitative Real-time PCR.The results indicated that the expression of this gene changed during the time course of the proliferation phase,and the PMC expression was detected at 5-month,but was slightly up-regulated before it reached the highest point at 8-month age.This change of PMC expression was corresponding with the phenomenon of ovaries at cellular level,in which additional replication of chromosomes occurred.These results could provide a clue to understand the molecular cheminism of diploidy hybrids producing diploidy eggs.