Molecular Cloning And Expression Analysis Of Four Different DnaJ Genes From Macrobrachium Rosenbergii

The DnaJ proteins are members of Hsp40 family which present in a variety of biological cells,they are ubiquitous stress response factors.They are originally found in Escherichia coli.DnaJ proteins play important roles,alone or in association with Hsp70 partners,in protein folding,refolding and protein complex stabilization under stress conditions.Macrobrachium rosenbergii belongs to Arthropoda,Crustacea,Decapoda,Palaemonidae,Macrobrachium.It is found throughout the tropical and subtropical areas of the Indo-Pacific region.Macrobrachium rosenbergii is considered a freshwater species.The giant river prawn has also been introduced to China.Its farming has dramatically increased within the last decade when shrimp culture suffered a major setback due to repeated attack by viruses.In this study,four DnaJ protein genes DnaJ A1,DnaJ B6,DnaJ C2 and DnaJ C5 were cloned from Macrobrachium rosenbergii.And their structures are analyzed and classified.The expression of DnaJ was studied in different tissues,under temperature stress and bacterial stress.The major results are as follows:The total RNA were extracted from the liver of Macrobrachium rosenbergii(L-C)and macrobrachium rosenbergii after Aeromonas hydrophila infection(L-I),then the transcriptome libraries of Macrobrachium rosenbergii were constructed by useing illumina Hiseq platform,and the transcriptome data were analyzed by bioinformatics methods,such as sequencing assessand gene annotation.The results showed that a total of 46800 contigs,from which 37605 Unigenes with an average length of 1073 bp were defined.19382 Unigenes were annotated.This research enriched the Macrobrachium rosenbergii gene resources.According to the results obtained from the transcriptome sequencing,we screened the genes which were significantly different before and after the infection of Aeromonas hydrophila,and finally determined the DnaJ gene.Then we designed RACE specific primers according to the known fragments,and obtained the full length of cDNA,The full lengths of Dna J A1,DnaJ B6,DnaJ C2 and DnaJ C5 were 1358,999,2319 and 995 bp.Comparison of DnaJ genes between Macrobrachium rosenbergii DnaJ and other species,and this four DnaJ genes were classified according to the result of the comparison.Dna J A1 should belong to Type I DnaJ subfamily as a result of existence of all four domains,J domain,C/F domain,CR domain and CTD.DnaJ B6 belongs to Type II DnaJ subfamily due to lack of the CR domain.DnaJ C2 and DnaJ C5 belong to Type ? DnaJ subfamily due to possess only the J domain.Total cell RNA was extracted from different tissues of healthy Macrobrachium rosenbergii,cDNA was synthesized for fluorescence quantitative PCR.The results of qRT-PCR showed that DnaJ A1,DnaJ B6,DnaJC2 and DnaJ C5 genes were expressed in six tissues and were significantly expressed in ovarian tissues(P <0.05),But in other tissues were different.The temperature stress has chosen four gradients: 15 ?,20 ?,25 ?,30 ?.At these temperatures,1 h was induced,and then the total RNA was extracted from muscle and liver,and cDNA was synthesized for fluorescence quantitative PCR.The results of qRT-PCR showed that four different DnaJ genes of Macrobrachium rosenbergii were expressed in liver and muscle at 15 ?,20 ?,25 ? and 30 ?.These four DnaJ genes were sensitive to cold stress in the liver and were significantly expressed at 15 ? and 20 ?(P <0.05),which had a certain stress effect on cold stress.However,the expression in muscle was different,such as Dna J A1,which was significantly lower(P<0.05)at low temperature.DnaJ B6 showed a down-regulation at 15 ?,20 ? and 30 ?,which indicated that low temperature stress and high temperature stress could reduce the expression of DnaJ B6.There was no significant difference in the expression of DnaJ C2 at four temperatures(P > 0.05).The expression of DnaJ C5 at 15 ? was significant(P < 0.05).This may be related to their structural differences.In order to understand the role of four DnaJ genes in bacterial defense,bacterial infection experiments were carried out with Aeromonas hydrophila.The liver tissues were taken at 0,1,3,6,9,16 and 24 h after infection with Aeromonas hydrophila.Total RNA was extracted and reverse transcribed into cDNA for fluorescence quantitative PCR.The results showed that the expression of DnaJ genes were significantly up-regulated(P < 0.05)after bacterial stress,which indicated that these four DnaJ genes might be involved in the bacterial immune response of Macrobrachium rosenbergii.But they also have some differences.For example,after 1 h infection,DnaJ A1 and DnaJ C2 were highly expressed(P<0.05).The DnaJ B6 and DnaJ C5 were significantly expressed at 3 h and 6 h(P<0.05)and peaked at 6 h.Moreover,the expression of DnaJ A1 and DnaJ C2 were significantly higher than that of DnaJ B6 and Dna J C5 after the Aeromonas hydrophila infection.The structure difference might account for their functional differences.