| Portunus trituberculatus is one of the most important marine cultured crabs in China.There are significant differences in phenotypic characters and economic value between male and female individuals.Therefore,the establishment of sex control technology and the realization of monoculture of Portunus tritub erculatus can significantly improve economic benefits.The selection of sex related markers and genes and the analysis of sex determination mechanism are the necessary prerequisites for the establishment of sex manipulation technology of Portunus trituberculatus.On the basis of previous work,this study screened and verified a molecular marker closely linked to the sex of Portunus tritub er culatus,established an efficient genetic sex identification technology,evaluated the sex ratio(sex ratio)of Portunus tritub erculatus in its early development stage,and anchored an important sex related gene doublesex.The results provide an important basis for the establishment of sex control technology of Portunus trituberculatus,and help to analyze the sex determination mechanism of Portunus trituberculatus.14-3-3 gene is a highly conserved gene,which is involved in many physiological processes such as cell stress,immunity,cell cycle regulation,cell signal transduction,metabolic enzyme synthesis,cell apoptosis and so on.It has been cloned in many economic crustaceans and preliminarily proved to play an important role in the process of resistance and disease resistance.However,up to now,there is no report about this gene in Portunus trituberculatus.In this study,we cloned the full length of 14-3-3 gene of Portunus trituberculatus,analyzed its expression pattern in the main tissues,studied its expression pattern after low salt stress and different pathogen infection,in order to explore the function of pt14-3-3 in salinity adaptation and immune response of Portunus trituberculatusThe research content of this paper includes the following four aspects:1.Screening and verification of sex linked molecular markers in Portunus trituberculatusBased on the results of sex QTL mapping of Portunus trituberculatus.a molecular marker marker3840 linked to sex 100%in mapping family was screened.By using the method of monoclonal sequencing,it was confirmed that the marker was an indel marker with 17 BP base difference.In order to test whether the marker can also be used for sex identification in other families and populations,this study used PCR product sequencing method to mark marker 3840 in two full sib materials(20 males and 20 females)and two population materials(60 females and 59 males).The results showed that the marker could be used to identify sex in 100%of the family materials and 98%of the population materials.At the same time,we found that the marker was heterozygous in male,homozygous in female,and supported Portunus trituberculatus as XY sex determining species,which was consistent with our previous research results2.Establishment of sex identification technique and analysis of the sex ratio of the whole sib of Portunus trituberculatusAccording to the Marker3840 marker characteristics,a pair of PCR primers that can amplify 101bp products were designed on the basis of 17bp base aberration point,and a 4%gender agarose gel electrophoresis was used to establish a sex identification technique for Portunus trituberculatus.The technique can be used to identify the genetic gender accurately and quickly by only one step PCR and agarose gel electrophoresis.The results are clearly visible,while the male crab strips show double bands while the female bands show a single band.Compared with PCR product sequencing and typing technology,this method is simple in operation and high in success rate,which greatly improves the identification efficiency and reduces the cost.Using this technique,sex ratio analysis of 7 whole sib families(Phenotype does not show sex differentiation)was carried out.The results showed that sex ratio of 3 families deviated significantly from 1:1(1:2.429;1:2.133;1:2).The results showed that the ratio of male to female of the three families was close to 1:1 at the stage of M and Z4,and there was no significant segregation(P>0.05).The results showed that the ratio of larval stage to larval stage of Portunus trituberculatus was 1:1,which was consistent with the theoretical ratio of sex determining type XY.Some families have sex ratio deviation of 1:1 in the second stage of juvenile crab,It is speculated that it may be due to the excessive density in seedling stage,which may lead to fighting among individuals and eating disability3.Cloning and expression analysis of double sex gene of Portunus trituberculatusThe full-length cDNA sequence of double sex gene of Portunus trituberculatus was cloned,which is 2484 BP long,798 BP open reading frame(ORF)and encodes 265 amino acids.The results of phylogenetic tree analysis showed that the gene was first clustered into one diplesex gene of Litopenaeus vannamei,and then into one diplesex gene with Oriental spiny lobster and Macrobrachium rosenbergii.PCR amplification of the gene was carried out in the DNA materials of families(20 adult females and 20 adult males)and populations(60 adult females and 59 adult males).It was found that the gene could only be amplified in males,indicating that it was closely linked with sex and might be a male specific gene.The expression of doublesex gene in different developmental stages of embryo and larva was studied by real-time fluorescence quantitative PCR.The results showed that the expression of doublesex gene in heart beat stage and Z3 was higher than that in blastocyst stage(18.54 times and 6.20 times,respectively)(P<0.05)(the lowest expression in blastocyst stage).It was speculated that these two stages might be the key period of sex differentiation of Portunus trituberculatus4.Cloning of 14-3-3 gene of Portunus trituberculatus and analysis of its expression after low salt and pathogen stressIn this study,the 14-3-3 gene of the swimming crab was cloned using the Rapid Amplification of cDNA Ends(RACE)method.The full length was 2510 bp and the ORF was 741 bp,encoding 247 amino acids and with a predicted molecular weight of 27.98 kDa.Sequence alignment analysis showed that the Pt14-3-3 gene has the highest homology with the 14-3-3 gene of Eriocheir sinensis(100%).Phylogenetic tree analysis indicated that the Pt14-3-3 amino acid sequence of P.trituberculatus was closely clustered into one with Eriocheir sinensis.Tissue expression analysis showed that the Pt14-3-3 gene was expressed in hepatopancreas,muscle,gill,heart,eyestalk,and hemolymph,and the expression level was highest in hepatopancreas.After low salt stress,the expression levels of the Pt14-3-3 gene in sputum and hepatopancreas were significantly upregulated at 48 h and 12 h,respectively,and reached the maximum,which was 1.34 times(P<0.05)and 7.54 times(P<0.05),respectively.After artificial infection with Vibrio parahaemolyticus and WSSV,the Pt14-3-3 gene was upregulated in the hepatopancreas and blood cells and the lowest was up to 17.52 times(P<0.05).The results of this study indicated that the Pt14-3-3 gene plays an important role in low salt adaptation and immune response of the Portunus trituberculatus. |
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