| Takifugu rubripes is an economic species in northern aquaculture.In order to improve the quality of germplasm and reduce the economic losses,we did some research about genealogical identification,cold tolerance gene screening and low temperature related regulation mechanism.We did experiment by using SSR,q PCR and transcriptome data analysis in Takifugu rubripes breeding groups.We choosed the liver tissues at the temperture of 18?,8? and 5? for transcriptome sequencing,we studied the different genes through the KEGG database by pathway analysis,the results showed that the differences were mainly concentrated in the metabolic pathway,and there were more differences in signal transduction,transport and metabolism.According the significant enrichment analysis of GO function,the results showed that with the decrease of temperature,some of the digestive enzyme activities related to molecular function were downgraded to different degrees,which to some extent on the fish's own body's metabolic intensity had a related impact,enzyme activity and regulatory mechanisms of metabolism,signal transduction and so has an important contact function.In the process of temperature drop,the binding of iron and the binding of heme also showed a downward trend,which was inextricably linked with the decline of fish activity in low temperature environment.Under the low temperature stress,the fish activity weakened,the breathing slowed down,and the oxygen inhaled inside the fish body also decreased.The dissolved oxygen condition of the whole fish also played a certain regulation to the fish body itself.At the low temperature,Most of the energy as a heat to balance the internal environment and the outside world caused by the temperature difference between the temperature,gill breathing weakened,so that the oxygen in the water into the blood less chance of the body cells are damaged,the cell membrane permeability becomes larger So that the external ions into the body,making the iron ion binding capacity decreased.With the decrease of temperature,DNA binding,RNA binding,chromatin binding,transcription factor binding,protein arginine methyltransferase(PRMTs)and core promoter sequence-related regulation are on the rise,we thought it is closely related to the regulation of the function of some proteins,such as protein arginine methyltransferase has been found in the regulation of chromatin structure,protein splicing and assembly,DNA repair,transcription regulation,signal transduction and other aspects of play an important role.In order to study the low temperature mechanism of Takifugu rubripes,the expression of gene antifreeze protein(AFP),Cold inducible RNA-binding protein(CIRP),high speed migration protein family protein(HMGB1),and Y-box binding protein in the liver,spleen,kidney,brain,heart,intestine,muscle,gonad and skin of Takifugu rubripes at the temperature of 18?,13?,8? and 5?.Studies have shown that four genes were expressed in each tissue,The expression level of AFP gene in each tissue was increased at 5? which was significantly higher than control group(P <0.05);In heart,intestine and skin tissues,The expression level of CIRP gene was the highest when the temperature was decreased to 8?,and the expression level of CRIP gene was the lowest at 5?(P <0.05).In the intestine,the expression of CRIP gene was significantly higher than control group at the temperature of 13 ?,8 ? and 5 ?(P <0.05).In the spleen,muscle and gonad,the expression of CIRP gene showed an increasing trend with the decrease of temperature,and the maximum expression level of CIRP gene at 5 ? was significantly different from control group(P <0.05).The expression of HMGB1 gene in each tissue was significantly increased,and the expression of HMGB1 gene in the spleen,kidney,brain,heart,intestine and gonad was significantly different from control group(P <0.05),and the expression level of HMGB1 gene was the highest when the temperature was decreased to 8?,the expression of HMGB1 in each tissue decreased when the temperature decreased from 8 ? to 5 ?.The Y-box gene was expressed in each tissue at different temperatures.In the spleen,intestine and gonad,the expression of Y-box was significantly higher than control group at the temperature of 5?(P <0.05).In muscle,The expression of Y-box was significantly higher than that in control group at the temperature of 8?(P <0.05).In spleen,kidney,brain,intestine and gonad,the expression of Y-box gene was significantly higher than control group at the temperature of 13?(P <0.05).These result suggests that AFP1 gene and HMGB1 were changed regularly with temperature.It shows that AFP1 gene and HMGB1 gene play an important role in the regulation of cold stimulation in Takifugu rubripes.Though CIRP gene and Y-box gene were expressed,there are not obvious change regulation,maybe there have a close relationship with the mechanism of the response mechanism under the low temperature stress.The transcriptome data shows that some of the digestive enzymes involved in molecular function were downgraded to different degrees with the decrease of temperature,and the binding of iron ions and the binding capacity of heme were also decreased when the temperature decreased.But the regulation of protein-related regulation showed an increasing trend with the decrease of temperature,it indicated that the mechanism of metabolism,immunity and regulation of Takifugu obscurus played an important role under the low temperature stress.In order to study the genetic diversity and genealogical information of Takifugu obscurus,then providing the theory of genetic improvement,five families were randomly sampled from twelve families which have been established in2015.Eight pairs of microsatellite primers were used to identify the genealogy and analyze the genetic diversity among five cultured populations of Takifugu rubripes.Thirty-two alleles were detected at eight microsatellite loci.The number of alleles(A)was 3 ~ 6 with an average of 4.The value of average observed heterozygosities was 0.4533 ~ 0.9533.The expected heterozygosities(He)was 0.4908 ~ 0.7901 and the mean polymorphic information content(PIC)ranged from 0.4555 to0.7546.Eight family unique alleles were found out: three in 7# family,two in 3# family,two in 11# family,one in 12# family.Family unique allele was not found in family 10#.Based on the genotypes of offspring,all parental genotypes of the five families were successfully deduced.3# family and 11# family was identified from the other families at locus fms15.7# family was identified from the other families at locus TOG01.12# family was disgusted from the other families at locus f1497.the three microsatellite markers(fms 15,TOG01,f1497)could be used to identify five families.7 pairs of microsatellite primers were used to identify the genealogy and analyze the genetic diversity between four cultured populations of Takifugu obscurus.Twenty nine alleles were detected at seven microsatellite loci.The number of alleles(A)was 3 ~ 6 with an average of 4.1.The value of average observed heterozygosities was 0.4917 ~ 0.9583.The expected heterozygosities(He)was 0.5270 ~ 0.7691 and the mean polymorphic information content(PIC)ranged from 0.4669 ~ 0.7272.Three family unique alleles were found out: two in 6# family,one in 12# family.Family unique allele was not found in family 11# and 14#.Based on the genotypes of offspring,all parental genotypes of the four family were successfully deduced.6# family was identified from the other families at locus f362 or f169.12# family was identified from the other families at locus f1372.11# family was disgusted from 14# family at locus f1372.f169 and f1372,f362 and f1372 the two pairs of microsatellite markers could be used to identify four families. |
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