Study On The Structure And Expression Of Spo-11 In Hybrids Of Carassius Auratus Red Var.(?)×Cyprinus Carpio L.(?)

In the process of distant hybridization of fish,the inheritance and expression of the key genes controlling meiosis in the formation of hybrid fish influence directly whether meiotic behavior of the germ cells occurs normally in the gonad,and obtain the ability to overcome the reproductive disorder.Spo-11 is one of the major genetic marker genes in the pre-meiotic phase,it encode a topoisomerase recognizing the site where DNA breaks to produce DSBs?Double Strand Breaks,related to chromosome recombination and repair in meiosis I?.Its normal expression is an important prerequisite for the normal occurrence of chromosomal behavior including homologous recombination.In this research,we choose the hybrids of Carassius auratus red var.???×Cyprinus carpio L.???as experimental subjects,of which production can represent the genetic characteristics of fish distant hybridization.We have obtained the cDNA and DNA samples of experimental fishes including common red crucian carp?RCC?,xiangjiang wild carp?CC?,allodiploid hybrid F₁?F₁?,allotetraploid progeny?4nAT?.And then analysis the homology of cDNA and some introns sequence of Spo-11 to know the genetic change of Spo-11 in the hybridization.Collect the testis from these four kinds of fishes living in the early breeding season,analysis the relative expression of mRNA and protein of Spo-11in these testis,watch the expression of SPO-11 protein in their germ cells to know the expression change of Spo-11 in the hybridization.We do this research to preliminarily explore the inheritance and expression patterns of key genes controlling the meiosis in the fish distant hybridization.Besides,we hope to obtain the relevant molecular biological evidence which can prove the hybrid fish overcome the reproductive disorders.The experiment results are as follows:?1?The CDS sequences of Spo-11 in RCC and CC both are 1152 bp in length,and the homology between them is only 95%.Besides,in the protein coding effect,RCC has two different mutant forms of coding sequences;CC has only one form;F₁ has four forms;4nAT has one form.In the genetic way,there are three different patterns:one is inherited from the female parent-RCC,another is inherited from the male parent-CC,and the other type is produced by parental genetic recombination.Currently,only one CDS sequence produced by genetic recombination of parents is detected in 4nAT which is more biased to RCC.?2?According to the homologous of sequences of the same introns from these all four kinds of experimental fishes,the sequences of whether intron-6 or intron-10 of Spo-11 are different stably in RCC and CC.Each sample of F₁ is inherited heterozygously with an intron from RCC and the other intron from CC,and the heterozygosity is inherited stably to 4nAT,and mutations such as base substitutions and deletions have occurred in the hybridization.?3?The Spo-11 in the testis of each experimental fish is transcribed and translated successfully.Histological results show that the Protein SPO-11 is mainly expressed in primary spermatocytes,but the expression level is low or almost not expressed in other germ cells.The relationship among the proportion of primary spermatocytes and the relative expression level of mRNA and protein of Spo-11 in the testis is positively correlated.Interestingly,the types of Protein SPO-11 expressed by RCC,CC and 4nAT are consistent with the types of Spo-11 mRNA coding sequences,but the four types of coding sequences of F₁ express only one type of protein.The function of Spo-11 in regulating meiosis of hybrid fish remains to be further studied.In summary,although the Spo-11 gene structures of different kinds of fishes in the hybridization have undergone different forms of mutation,all experimental fishes have expressed Protein SPO-11 normally.The results preliminarily prove that the functional expression of meiotic key genes is normal during the formation of hybrid fish,which build the molecular foundation for hybrid fish to overcome reproductive obstacles.