| Polyploidy is an important feature of species evolution.Distant hybridization is an effective way to transfer the genome of one specie to another,for which the hybrids showed heterosis in phenotype and genotype in polyploid formation.The F2 generations between the distant hybridization of red crucian carp(Carassius auratus red var.,RCC,♀)and common carp(Cyprinus carpio,CC,♂)were found that produce unreduced diploid gametes,self-crossing of F2 resulted in the appearance of allotetraploid hybrids(4nAT)in F3,and we have obtained the genetic stable and fertile tetraploid fish line(F3-F27).The distant hybridization between the female RCC with the male allotetraploids were obtained the sterile allotriploid crucian carp.The sterile triploid fish has strong disease resistance and growth fast.Because of the sterile triploid fish gonad is abortion,it is widely cultivated in China because it has the advantage of no influence for the natural resources in any water.Dnah3 is relating to the flagella assembly of spermatozoa,the movement of flagella and the activity of spermatozoa,Dnah3 is a kind of axonemal dynein heavy chain.Ift57 is essential to maintain the length of the flagella and the two-way material transport.Abnormal mutations of these two genes can lead to the abnormal of flagella,and then result in the failure to produce normal sperm.In the previous study,we have successfully constructed the miRNAs library in different ploidy cyprinids,we found that Dnah3 and Ift57 were the target genes of miR-199-5p and raiR-221,respectively.The partial cDNA sequences of Dnah3 gene and Ift57 gene were cloned,and the expression of these two genes in different ploidy Cyprinids was studied.microRNA is a kind of endogenous non-coding small molecule RNA with a length of about 17-24 nt in eukaryotes.miRNA regulates the expression of genes through the complete or incomplete complementation of the 3’ UTR region of the target gene.More and more research showed that miRNA is closely related to organ formation,embryonic development,tumorigenesis,cell proliferation,differentiation and apoptosis.In this paper,we constructed the miRNAs library of diploid RCC and 4nAT in ovaries tissues,in addition,bioinformatics analysis was carried out in several aspects,and the results were verified by experiments.From the miRNA level,the propagation characteristics of tetraploid fish lines formed by distant hybridization were expounded.1.The partial cDNA sequences of Dnah3 gene of diploid red crucian carp,triploid crucian carp and allotetraploid crucian-carp were cloned and analyzed,and the expression of Dnah3 gene of different ploidy cyprinids in 9 kinds of tissues was studied and analysed,the results showed that the expression in testicle was higher than in other tissues.At the same time,we through the qPCR to studied the expression of Dnahi gene of different ploidy cyprinids in testis tissues,the qPCR results showed that the expression of Dnah3 in triploid was lower than diploid and tetraploid.2.The partial cDNA sequences of Ift57 gene of diploid red crucian carp,triploid crucian carp and allotetraploid crucian carp were cloned and analyzed,and the expression of Ift57 gene of different ploidy Cyprinids in 9 kinds of tissues was studied and analysed,the results showed that the expression in testicle was higher than in other tissues.At the same time,we through the qPCR to studied the expression of Ift57 gene of different ploidy cyprinids in testis tissues,the qPCR results showed that the expression of Ift57 in triploid was lower than diploid and tetraploid.3.In this experiment,we collected three ovary tissues from RCC and 4nAT respectively,and constructed small RNA library with high-throughput sequencing.After the screening of the raw data we obtained the clean reads,the data are as follow:1,252,130(HJ1♀),13,885,093(HJ2♀f),10,318,700(HJ♀f),12,888,037(4nl♀f),13,012,243(4n2♀f),11,342,865(4n♀f).We mapped the small RNA sequences to the reference genome through the MIRDeep2 software,the results showed that there were 441 mature miRNAs sequences in the diploid red crucian carp and 418 mature miRNAs sequences were discovered in 4nAT.Compared with RCC there were 9 conserved miRNAs expression were down regulated in 4nAT,and 30 conserved miRNAs and 4 non-conserved miRNAs were up regulated.Based on the high-throughput sequencing result,we selected 5 different expressed miRNAs(miR-6843-3p,miR-81b-p,miR-42-5p,miR-21-5p and miR-2368-3p)in different ploidy fish ovary,then through the quantitative real-time PCR to test and verify the relative expression level of these 5 different expressed miRNAs.The result of qRT-PCR was consistent with the result of high-throughput sequencing.Then we predicted target genes sequence of obvious different expressed miRNA in the miRanda database,we also annotated and classified the function of target genes through blast against with the NR,Swiss-Prot,GO,COG and KEGG database. |
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