Study On The Inheritance And Expression Of Mlh1 Gene In Hybrid Fish From Carassius Auratus Red Var.(Female) X Cyprinus Carpio(Male)

The repair mechanism of meiotic mismatch played an important role in maintaining normal meiosis after mismatches occurred in the formation of heterologous double stranded DNA during meiotic recombination.Mlh1 gene was involved in the recombination and crossover of homologous chromosomes during the first meiotic division,and was an important marker factor for studying the characteristics of meiosis.The meiosis process of distant hybrid fish with heterozygotes was a matter of concern.In this study,the genetic and expression characteristics of Mlh1 in Carassius auratus red var,Cyprinus carpio,F₁of Carassius auratus red var and Cyprinus carpio and allotetraploid and so on,were studied by molecular cloning,Q-PCR,Western Blot and immunohistochemical methods,and the relationship between Mlh1 and the original parents was analyzed.The results and conclusions were as follows:?1?The complete CDS sequences of RCC and CC Mlh1 were2175bp and 2172bp respectively.The most significant difference of CDS sequences between RCC and CC was whether there was TAGTTCCTC or not.The results of sequence alignment showed that there was a stable difference between the original RCC and CC.CDS sequences of different types of Mlh1 genes were obtained for both F₁ and 4nAT.According to whether there were TAGTTCCTC fragments,the CDS sequences were divided into 2175bp?F₁-1,4nAT-1?of RCC and 2172bp?F₁-2,4nAT-2?of CC.However,the genetic characteristics of offspring RCC and CC were not identical with their parents,and there were some genetic variations,and the genetic variation loci with irregularly and randomly.?2?According to the CDS sequences of Mlh1 genes of four fish species,primers of the common conservative region of Mlh1 genes were designed to clone and amplify the DNA sequences of Mlh1 genes of female RCC,male CC and hybrid strain of RCC and CC,and multiple fragments of Mlh1 genes were obtained and spliced.The partial length of Mlh1 DNA splicing of RCC was 4696bp,including 16 exon sequences.The partial length of Mlh1 DNA splicing of CC was 4106bp,including 13exon sequences.The DNA sequences of Mlh1 genes of F₁ and 4nAT both had two types:the partial splicing lengths of RCC which contained 16exons and the partial splicing lengths of CC which contained 13 exons respectively.Preliminary analysis of some intron and exon sequences of Mlh1 genes of four fish species showed that both Mlh1 genes of F₁ and4nAT were two types:type of RCC and type of CC,and no gene chimerism was found but genetic variation existed.?3?The gene expression of 4nAT in the same season was 1.307 times higher than that of RCC Mlh1 in the same season,while that of CC and F₁in the same period was 4.988 and 16.652 times higher than that of RCC Mlh1 in the same season,respectively.The results showed that the difference was positively correlated with the proportion of primary spermatocyte.When the proportion of primary spermatocyte was high,the expression of Mlh1 gene was high too.It was concluded that a large number of spermatogenesis of F₁ were blocked in the primary spermatocyte stage.?4?The relative abundant expression of Mlh1 protein in primary spermatocyte was detected by Western Blot and immunohistochemistry,which could be used as a kind of protein marker to further study the germ cell development characteristics of hybrid fish.This study was the first to analyze the genetic characteristics and related functional parameters of key meiotic genes in distant hybrid fish,which was of great significance to the genetic evolution of distant hybrid fish and the practice of fish genetics and breeding.