| The Chinese shrimp,Fenneropenaeus chinensis,belonging to Arthropoda,Crustacea,Decapoda,Natantia,Penaeidae,Fenneropenaeus,is a large-scale migratory shrimp with wide temperature,wide salinity and annual warm water.It has the characteristics of strong adaptability,fast growth,low temperature tolerance,nutrient-rich and long-distance migration.It is an important farmed aquatic species in China.F.chinensis farming has a wide range,mainly distributed in the coastal areas of the Yellow Sea and the Bohai Sea north of the Yangtze River in China.Due to the soil characteristics of coastal areas,the pH of seawater in F.chinensis culture areas is generally high,and the pH value is between 8.5 and 9.5 for a long time.In the hot summer months,the pH of the sea is even higher than 9.5 due to photosynthesis.Compared with Penaeus vannamei and Marsupenaeus japonicus,F.chinensis are less tolerant to pH.Higher pH is extremely unfavorable for the growth and breeding of F.chinensis,which has become an important factor restricting the large-scale cultivation of F.chinensis.V-ATPase is an important pH stress response enzyme,which plays an important role in the corresponding process of animal and plant stress resistance,but it has not been reported in F.chinensis.In this study,we investigated the genetic response of V-ATPase at high pH in order to understand the regulation mechanism of V-ATPase in the resistance to pH stress of F.chinensis and provide a theoretical reference for the breeding of stress-resistant varieties of F.chinensis.The research content includes the following three parts: 1.Cloning and Sequence Analysis of V-ATPase c,c? and e Subunits in in F.chinensisThe full-length cDNA sequence of V-ATPase c subunit,c? subunit and e subunit gene of F.chinensis were cloned by rapid amplification of cDNA ends(RACE),and then the sequences were analyzed.The results showed that the full length cDNA sequence of V-ATPase c subunit gene in F.chinensis was 2128 bp,the length of open reading frame was 483 bp,the 5' non-coding region and the 3' non-coding region contained 105 bp and 1540 bp,respectively.The gene was named FcVHA-c,speculated that the gene encodes 160 amino acids,predicted protein molecular weight of 16 kDa,theoretical isoelectric point of 7.82,is a hydrophobic multiple transmembrane protein,including four transmembrane domains: TM1~TM4,TM4 contains a glutamic acid residue Base site.Homology analysis revealed that the amnio acid sequences of FcVHA-c had highly identifies with P.vannamei,which was 99%,and the phylogenetic analysis showed that F.chinensis was in the same class with P.vannamei first and then Hyalella Azteca.The full length cDNA sequence of V-ATPase c?? subunit gene in F.chinensis was 1649 bp,the open reading frame included 633 bp,the 5' non-coding region and the 3' non-coding region were 101 bp and 915 bp,respectively.The gene was named FcVHA-c??.The gene was predicted to encode 210 amino acids with a molecular weight of 21 kDa and a theoretical isoelectric point of 5.09.It is a hydrophobic multiple transmembrane protein,including five transmembrane domains: TM0~TM4,and TM4 contains a glutamic acid residue Base site as well.Homology analysis revealed that the amnio acid sequences of FcVHA-c?? had highly identifies with P.vannamei,which was 98.57%,and the phylogenetic analysis showed that F.chinensis was in the same class with P.vannamei first.The full length cDNA sequence of V-ATPase e subunit gene in F.chinensis was 546 bp,the open reading frame included 246 bp,the 5' non-coding region and the 3' non-coding region are 32 bp and 268 bp,respectively.The gene was named FcVHA-e.The gene encodes 81 amino acids,and the predicted protein has a molecular weight of 8.8 kDa and a theoretical isoelectric point of 8.63.It is a hydrophobic transmembrane protein that crosses the membrane one time.Homology analysis revealed that the amnio acid sequences of FcVHA-e had highly identifies with Diaphorina citri,which was 67%,and the phylogenetic analysis showed that F.chinensis was in the same class with P.vannamei first.2.Tissue expression analysis of V-ATPase c subunit,c? subunit and e subunit genes in F.chinensis and their expression in gills under high pH stressFluorescence quantitative PCR was used to detect the expression distribution of V-ATPase c subunit,c? subunit and e subunit in various tissues of F.chinensis and the expression of each gene in gills under high pH stress.The results showed that the FcVHA-c,FcVHA-c? and FcVHA-e gene were expressed in all tissues.FcVHA-c gene had the highest expression in gill,14.557 times as much as in heart,followed by eyestalk,hemo lymphocyte and intestine,and the lowest expression in heart.FcVHA-c? gene had the highest expression in gill,51.328 times as much as in heart,followed by hemo lymphocyte,eyestalk and antennary gland,and the lowest expression in heart.The expression level of FcVHA-e gene in gill was significantly higher than that in other tissues(P<0.05),93.972 times as much as in eyeball,followed by higher expression in eyestalk,hepatopancreas and intestine,and the lowest expression in eyeball.FcVHA-c,FcVHA-c? and FcVHA-e genes were most expressed in gills,suggesting that V-ATPase was mainly distributed in gills.Under pH 8.8 stress,the expression of FcVHA-c,FcVHA-c? and FcVHA-e genes in gills increased firstly and then decreased with the stress time.After pH stress,the expression of three genes reach the highest value at 12h?12h and 24 h,respectively,with 1.206,1.574 and 1.790 times as compared with the control group,respectively.Under the stress of pH 9.2,the expressions of FcVHA-c and FcVHA-e genes increased firstly and then decreased,reaching the highest value at 1h and 24 h after stress,respectively,which was 1.577 times and 3.173 times higher than that of the control group.Under the stress of pH 9.2,the expression of FcVHA-c " gene first increased,then decreased and then increased with the stress time,reaching the peak at 1h and 48 h,which was 2.834 times and 2.384 times of the control group,respectively.The above results indicated that FcVHA-c,FcVHA-c" and FcVHA-e gene play important regulatory roles in response to high pH stress,and different subunits respond differently to high pH stress and time.3.Functional study of V-ATPase in F.chinensis under high pH stressThe siRNA interference technique was first used to analyze the function of V-ATPase in F.chinensis.Under high pH stress,the FcVHA-c gene and FcVHA-c? gene were interfered respectively.The mortality rate of FcVHA-c gene interference group was higher than that of FcVHA-c? group at 48 h than that of control group,which indicated that V-ATPase c and c" subunits could regulate acid-base balance under high pH stress.Under high pH stress,the gene expression of CAc,NHE3,AE,NBC and NKCC were detected after interfering the V-ATPase c subunit.The results showed that AE,NBC and NKCC genes were significantly up-regulated at 24 h after high pH stress,indicated that the deletion of c subunit might induce the positive response of AE,NBC and NKCC.It was speculated that AE,NBC,NKCC and V-ATPase had synergistic regulation under high pH stress environment.Inhibiting the activity of V-ATPase in F.chinensis,the mortality rate of the inhibition group was significantly higher than that of the control group after 48 hours under high pH stress(P<0.05),which indicated that V-ATPase participated in the regulation of acid-base balance of F.chinensis under high pH stress. |
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