| Rainbow trout(Oncorhynchus mykiss),which is affiliated to,Salmoniformes(Salmoniformes),salmon(Salmonidae),salmon genera(Oncorhychus)of the family of typical cold water farmed fish.The somatic cell growth of rainbow trout was significantly reduced when the rainbow trout was mature,and the energy was mostly used to satisfy the gonadal development and maintain the secondary sexual characteristics and reproductive behavior,which resulted in the degradation of the flesh and appearance quality of diploid rainbow trout.The oocyte development of triploid female rainbow trout was completely inhibited and the abnormal gonadal development eliminated the bad results caused by sexual maturity.However,the deep mechanism of abortion of triploid female rainbow trout glands is not well studied.On the basis of the preliminary work in our laboratory,this study continues to explore the molecular regulation mechanism of the developmental arrest of triploid rainbow trout oocytes.Because triploid female rainbow trout has three sets of chromosomes,in meiosis even line phase,that is,homologous chromosome association will occur disorder,so that meiosis can not proceed normally,unable to discharge mature gametes,resulting in no fertilization ability.In previous studies,Spindlin(Spin)gene has been proved to be a maternal effector factor related to meiosis spindles,and plays a role in cell cycle regulation during the transition from oocyte to early embryonic development.As a cytoskeletal structural substance,β-tubulin has the functions of maintaining egg cell morphology,coordinating intracellular material exchange and assisting cell proliferation during gonadal development.Both genes play important roles in sexual maturation.Therefore,this study related regulatory mechanism in meiosis as the breakthrough point,to select triploid female steelhead 240-330 dpf(days post fertilization,DPF)development period,Spindlin and β-tubulin gene cloning,using real-time fluorescent quantitative(RT-PCR),tissue stained with HE,western blotting method and immunohistochemical method to identify the two triploid female steelhead oocyte expression differences in different development stage of meiosis process conditions and two kinds of protein subcellular localization of different periods,Thus,the regulation of abnormal proliferation and differentiation of triploid female rainbow trout in sex cells was investigated.Specific research results are as follows:1.through rapid amplification cDNA end(RACE)technology,in 4,529 bp spindlin gene c DNA(Gen Bank login number: MN378564),named Os Spindlin.3’untranslated region(UTR)and 5’ untranslated region(UTR)were 3,662 bp and 141 bp respectively,open reading frame(ORF)was 726 bp,encoding 241 amino acids,the relative molecular weight of 28.3 KD protein sequences,theory of isoelectric point value of 5.94,not across the membrane structure.Homology analysis showed that rainbow trout(Oncorhynchus mykiss)and silver salmon(Oncorhynchus kisutch)had the highest homology,up to 99.59%.Phylogenetic trees show that rainbow trout clump together with salmon(Oncorhynchus tshawytscha)and salmon(Salvelinus alpinus).Spindlin gene was slightly expressed in diploid and triploid female rainbow trout at various stages of embryonic development,showing a decreasing trend,among which the expression level was the highest at fertilized egg stage.At the same developmental stage,the relative expression level of Os Spindlin gene in diploid rainbow trout was higher than that in triploid rainbow trout,and there were significant differences between diploid and triploid rainbow trout in fertilized egg stage,8-cell stage and tail bud stage(P<0.05),while there were no significant differences in other periods(P>0.05).Os Spindlin gene was expressed in ovary,kidney,liver,spleen,muscle,gill,heart,eye,intestine and fin tissues of adult rainbow trout,among which the expression level in ovary was significantly higher than that in other tissues(P<0.01).For female rainbow trout diploids,the relative expression of Os Spindlin gene in ovarian tissue decreased significantly during the development stage of 240-300 dpf after fertilization.For triploid female rainbow trout,the expression level of this gene increased significantly at the stage of 240-330 dpf.At the same developmental stage,the expression level of Os Spindlin gene in female rainbow trout diploid ovary was higher than that in female rainbow trout triploid ovary,and there were significant differences(P<0.01).Western blot results show that the Spin protein in 240-330 dpf stages are expressed,for diploid female rainbow trout,the highest amount of Spin protein expression in 240 dpf,decrease in 270 dpf.compared with diploid rainbow trout have the same change trend,triploid trout Spin in the same period,the diploid and triploid protein expression between have significant difference(P<0.05).Immunohistochemical results showed that the signal of Spin protein in the nucleus of primary egg was the strongest in the diploid of female rainbow trout at the stage of 240 dpf,and gradually weakened at the stage of270-330 dpf.Triploid rainbow trout ovaries develop at 240-330 dpf,and the signal in oocytes is gradually enhanced.The above studies showed that the abnormal occurrence of Os Spindlin gene in triploid female rainbow trout during meiosis was related to the low expression of spindlin gene.2.Rainbow trout β-tubulin gene open reading frame 1,533 bp was cloned,Gen Bank login number: MN931398 was obtained,and 424 amino acids were encoded.By analyzing the encoded protein,the relative molecular weight of steelhead β-Tubulin protein is 47.5KD,the theoretical isoelectric point value is 4.73,and the instability index is 38.85.This gene has no signal peptide and no transmembrane helical region.According to the comparison of amino acid homology of β-Tubulin,rainbow trout and silver salmon(Oncorhynchus kisutch)had the highest homology among bony fish(94.37%).Trout β-Tubulin system evolutionary tree results showed that the amino acid sequence and other salmonidae fish than for a alone,and the Atlantic salmon(salmo salar)and Chinook salmon(Oncorhynchus tshawytscha)of recently,genetic relationship,followed by basic is divided into two bony fishes and mammals,including genetic relationship with Gallus gallus,as far as other fish genetic relationship in between.The relative expression of β-tubulin gene in diploid and triploid female rainbow trout during embryonic development showed that the overall trend was first rising and then decreasing,and the highest expression was in the 2 cell phase,which was the fertilized egg,8 cell phase,and 16 cell phase.Compared with 28.4,7.2,7.9 and 9.5times of mulberry stage,compared with diploid rainbow trout,triploid rainbow trout had the same upward trend from fertilized eggs to 2 cell stage and there were significant differences(P <0.05).During the same development period,the expression level ofβ-tubulin gene in diploid rainbow trout was higher than that of triploid rainbow trout.β-tubulin gene is expressed in 10 tissues including fin,kidney,liver,spleen,muscle,gill,heart,eye,intestine,and ovary of diploid female rainbow trout.The expression in ovarian tissue was significantly higher than that in other tissues(P <0.01).By analyzing the relative expression levels of β-tubulin gene in different developmental stages of diploid female rainbow trout adult ovary tissues,during the 240-330 dpf development stage,the expression level first increased and then decreased,with the highest expression level at 270 dpf.It is 1.2,2.2 and 2.8 times of 240 dpf,300 dpf and 330 dpf.There was a significant difference in expression levels between 270 dpf and 240 dpf(P<0.05),and there was no significant difference between the expression levels at later stages(P>0.05).During the development stage of triploid rainbow trout at 240-330 dpf,the overall trend is the same as the relative expression of diploid rainbow trout ovary.At the stage of 240-270 dpf,the relative expression of β-tubulin gene significantly increased,with the highest expression at 270 dpf There is a very significant difference between 240 dpf and 270 dpf(P<0.01),but there is no significant difference between270-330 dpf(P>0.05).In general,in the same developmental stage of β-tubulin gene,the expression level of diploid rainbow trout is relatively higher than that of triploid rainbow trout.The expression and localization analysis of β-Tubulin protein in ovarian tissue showed that for diploid female rainbow trout ovaries,The gonadal expression trend ofβ-Tubulin protein and Spin protein was different at each stage.At the development stage of 240-330 dpf,the expression trend of β-Tubulin protein was first increased and then decreased,and the expression level reached the highest at the development stage of 270 dpf,while there was no significant difference in diploid(P>0.05).In other periods,there were significant differences between the diploid and triploid rainbow trout(P<0.05).Protein localization in ovarian tissue showed that,the positive signal was mainly concentrated in the cytoplasm at 240-330 dpf,and the signal showed a tendency of first strengthening and then weakening.his is different from the dynamic change of Spin protein.Compared with diploid female rainbow trout,triploid rainbow trout ovary was at 240-330 dpf stage,and the signals were mostly concentrated in follicular cells and supporting cells in the interstitial cell,while the signals on oblast cells were weak.The results showed that the abnormality of triploid rainbow trout eggs during proliferation and differentiation was related to the low expression of β-tubulin gene and the subcellular localization.According to the results of this experiment,it can be concluded that the disruption of oocyte maturation during the development of female rainbow trout’s gonads in triploid females may be related to the low expression of Os Spindlin and β-tubulin genes and the difference in the dynamic distribution of Spin and β-Tubulin proteins in the ovaries of diploid and triploid females,which provides strong evidence for further revealing the abortion of triploid gonads. |
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