Organisation And Expression Analysis Of 5S And 45S Ribosomal DNA Clusters In Autotetraploid Fish

Polyploidy is one of the main driving forces of biological evolution and can lead to dramatic changes in gene expression and structure.Due to its high copy rate in the genome and special conserved coding region arrangement,ribosomal DNA（rDNA）has become a hotspot in the study of repeated sequence molecular evolution.Autotetraploid carassius auratus（4nRR,4n=200）were derived from the whole genome replication of carassius auratus red var.（RCC,2n=100）,which had four sets of chromosomes from RCC and could generate reduced gametes stably（2n=100）,finally forming a genetically stable autotetraploid fish line(F₂-F₁₅).The genetic background of autotetraploid carassius auratus is clear,which provides valuable experimental materials for further understanding the evolution characteristics of rDNA in the process of polyploidy of fish.In this study,we studied the sequence,structure,methylation level and expression level of 5S rRNA and 45S rRNA genes in 4nRR,and revealed the effects of autotetraploidization on rDNA in fish.The main research contents and results were as follows:1)The sequences of 5S rRNA and 45S rRNA（5.8S,ITS1,ITS2）genes in 4nRR were compared and analyzed.The results showed that the cDNA sequences similarity of 5S rRNA gene coding region in 4nRR had two types:one that was completely consistent with RCC;although the other type had some nucleotide site variation,it had a high similarity with the control group in RCC（96.6%）.The similarity of 5.8S rRNA and ITS1 region were 100%.The results showed that two different types of ITS2 cDNA sequences（type Ⅰ,type Ⅱ）were found in 4nRR:type Ⅰ was the same with its diploid ancestor;type Ⅱ was with significant insertions,deletions and base substitutions.The results showed that 5S rRNA,5.8S rRNA and ITS1 region were highly conservative,while ITS2 region was differentiated after autotetraploidization.2)The secondary structures of 5S rRNA and 45S rRNA（5.8S,ITS1,ITS2）genes in 4nRR were predicted and analyzed.The study found that5S rRNA,ITS1,and 5.8S rRNA were highly conserved in sequences,and their secondary structures were relatively consistent between RCC and4nRR.The secondary structures showed that two types of ITS2 were functional,helix Ⅱ（pyrimidine-pyrimidine）and helix Ⅲ were highly conserved in the two types of secondary structures of ITS2,especially the5’side of the helix Ⅲ（CCGGTGG）.Helix Ⅳ was the most variable region in ITS2,there were differences between the two types of ITS2 in4nRR.The results suggested that autotetraploidization resulted in significant structural differentiation of ITS2 region,forming a new structural type with functions.3)The regulatory region methylation levels（NTS,IGS）and gene expression levels（5S,18S）of 5S rRNA and 45S rRNA in 4nRR were compared and analyzed.Compared with the diploid ancestor,there was no significant difference in the level of NTS methylation,but the expression level of 5S rRNA gene was lower in 4nRR,the results showed that the methylation of NTS region did not participate in the regulation of the expression of 5S rRNA in 4nRR.In comparison of IGS（45S rRNA）results,it was found that the level of methylation was higher in 4nRR,but the expression of 18S rRNA gene was not significantly different from that in RCC,indicating that methylation of the regulatory region was involved in the regulation of the expression of 18S rRNA gene after autotetraploidization.The above studies initially revealed the effect of autotetraploidization on the structure and function of 5S and 45S rRNA in carassius auratus,and provided a theoretical support for the systematic study of the evolution pattern and characteristics of ribosomal DNA（rDNA）in vertebrates.