| Exopalaemon carinicauda is an important small and medium-sized economic shrimp in China and mainly distributed in the shallow waters of the Yellow Sea and the Bohai Sea,E.carinicauda has becomed special culture species in coastal area of China with the advantages of fast growth,short reproductive period and strong adaptability to different environments.China has 46 million hm~2 of saline waters,of which 6.67 million hm~2 have potential for culture.Saline waters cannot be fully exploited because of their high p H,high carbonate alkalinity and complex ionic composition.E.carinicauda is an ideal material for conducting research on the molecular mechanisms of saline-alkali adaptation in crustaceans because of its strong adaptability to different environments.In this study,we estimated the heritability and genetic correlation of alkalinity tolerance traits in E.carinicauda by constructing family materials,screened key genes in the alkalinity tolerance mechanism combined with transcriptome,miRNA and SNP of E.carinicauda,and verified their functions in the process of alkalinity stress by RNA interference technology.The results provided basic information for selection process of E.carinicauda and revealed the alkalinity tolerance mechanism of crustaceans.The main research contents are as follows:1.Estimation of heritability and genetic correlation of alkalinity tolerance traits in E.carinicaudaThe heritability and genetic correlation of alkalinity tolerance traits in 50-day-old E.carinicauda were estimated using quantitative genetics principles and whole-sib group intra-association.Fifty full-sibling lines(including 42 half-sibling lines)of E.carinicauda were constructed using directed crosses.The heritability and genetic correlation of alkalinity tolerance traits were estimated by using the general linear model(GLM)of SPSS software to analyze the survival time of alkalinity stress in each line.The results showed that the heritability of alkalinity tolerance in E.carinicauda ranged from0.18 to 0.60,and the estimates did not reach a significant level,which was moderate;the genetic and phenotypic correlations of alkalinity tolerance with body length were-0.401and 0.127,respectively,and those of alkalinity tolerance and body weight were-0.196and 0.033.The results show that we can improve the alkali tolerance of E.carinicauda by selective breeding.In the breeding process,the growth traits and alkali-tolerant traits of E.carinicauda are comprehensively selected to cultivate new high-yield and stress-resistant varieties.2.Analysis of the alkalinity tolerance mechanism of E.carinicauda based on transcriptome,miRNA and SNPThe transcriptomic information of E.carinicauda under alkalinity stress were analyzed using high-throughput sequencing technology.A total of 136.15 Gb Clean Data were obtained,with the percentage of Q30 bases above 93.74%.14112 unigenes were assembled and 12,571 were successfully annotated,including 12,383 NR and 7,788KEGG.Quantitative analysis of genes and transcripts was performed using RESM software.Compared with the blank control,a total of 96 genes were differentially expressed after 24h of alkali stress,of which 56 genes were up-regulated and 40 genes were down-regulated;after 48 h of alkali stress,a total of 272 genes were differentially expressed,of which 147 genes were up-regulated and 125 genes were down-regulated.The differentially expressed genes were enriched by GO and analyzed for transport activity,substrate specific transporter activity,lipid transporter protein activity,and significant enrichment of molecular functions,which are closely associated with life activities such as transmembrane transport and ion transport.The results of miRNA sequencing in E.carinicauda after acute alkalinity stress revealed 20 differentially expressed miRNAs,six of which were newly identified,among which dme-miR-9a-5p and dme-miR-1-3p were both up-regulated during stress,which may be more closely associated with the alkalinity tolerance mechanism in E.carinicauda.In addition,we screened SNP closely linked to alkalinity tolerance in E.carinicauda based on the previous family materials.The high-quality SNP was analyzed by GWAS using mixed linear model,and 6 QTL associated with alkalinity tolerance were detected,and 85 SNP loci significantly associated with alkalinity tolerance were detected.These results provided basic information for investigating the mechanism of alkaline tolerance in E.carinicauda.3.Cloning and functional validation of the aquaporin 4 and 11 in E.carinicaudaBy analyzing the transcriptome sequencing data,we found that the expression of two isoforms of aquaporin(AQP):aquaporin4(AQP4)and aquaporin11(AQP11)were both changed under acute alkaline stress conditions.Aquaporin 4 and 11 gene were successfully cloned using RACE cloning technique.The ORF of AQP4 gene is 621bp,encoding 206 amino acids,with a predicted molecular weight of 21.673kDa and a theoretical isoelectric point of 8.30,and is a hydrophobic protein with five transmembrane structural domains.It is a hydrophobic protein with 4 transmembrane domains.Sequence alignment showed that AQP4 gene had the highest homology with Macrobrachium rosenbergii(94.63%),and AQP11 had the highest homology with Penaeus monodon(81.47%).In this study,RNA interference was used to silence the expression of AQP4and AQP11 genes to verify the function.The results showed that after the carbonate alkalinity stress,the mortality of E.carinicauda increased significantly after RNA interference injection.The mortality of AQP4 group reached 45%at 72h,and the mortality of AQP11 group reached 55%at 72h,which was significantly higher than control group.The blood osmotic pressure of the E.carinicauda in the AQP4 group was significantly higher than that of the control group;the blood osmotic pressure of the white shrimp in the AQP11 group was significantly increased.In summary,aquaporins play a role in regulating osmotic pressure and maintaining ion balance in the process of E.carinicauda responding to alkalinity stress. |
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