The Expression And Functional Analysis Of Heat Shock Protein 70, And 90 Family Genes In Dugesia Japonica

Dugesia japonica belongingto Platyhelminthes,Turbellaria,plays an important role in the evolution of animal systems and has a strong ability to regenerate.At present,although a large number of genes related to the regeneration of planarians have been cloned,the specific regeneration mechanism and the function of related genes are still obscure.In this study,the quantification,localization,and functional analysis of Djhsp70 e,Djhsp90a,Djhsp90 b and Djhsp90 c geneswere performed.These genes are of 70 and 90 families with continuously low expression in the regeneration process of Dugesia japonicafrom the transcriptome data.The spatial and temporal expression patterns and functions of four genes were analyzed by whole-mount in situ hybridization,RNA interference and Real time-PCR.The results are as follows:(1)The expression of four genes was analyzed by Real time-PCR technique under the conditions of cutting injury,ionic liquid exposure,cold stress and heat stress.The results showed that under the above conditions,the expression level of the four genes increased significantly.The expression of the four genes continuously increased as the length of the cutting and the concentration of the ions increased(2)The full-length cDNA ofDjhsp70 e gene was 1982 bp,the 180 bp noncoding region at the 5 'end and the 38 bp untranslated region at the 3' end,which contained a maximum open reading frame(ORF)of 1764 bp,encoding a polypeptide of 587 amino acids which displayed two heat shock protein 70 family-specific tag sequences.Whole-mount in situ hybridization showed that Djhsp70 e gene was widely expressed in the body except the brain and pharynx,and the hybridization signal on both sides of the body and the intestinal branch was stronger.The budding part of the regenerated individuals had strong hybridization signal.After interfering the gene,the whole planarians appear seriously head dissolved and significantly inhibited regeneration,and the first regeneration of the tail fragment also appears seriously dissolution phenomenon.Real time-PCR results showed that the expression of Djpiwi-1 and Djpiwi-b was significantly down-regulated after interfering with the gene.(3)The full-length cDNA ofDjhsp90 a gene of the genus Djhsp90 a was 3212 bp,including the noncoding region of the 5'-end 1039 bp and the non-coding region of the 3'nd end of the 22 bp,which contained an ORF of 2151 bp,encoding of a 716 amino acid protein.whichdisplayed six heat shock protein 90 family-specific sequence tags.Whole-mount in situ hybridization showed that the hybridization signals were mainly distributed in most of the normal tissues except the brain and pharynx.The buds in the regenerated individuals had strong hybridization signals.After interfering the gene,the whole headworm showed obviously head dissolution and accompanied by continuous shrinkage of the body.The regeneration of the head regeneration was inhibited.The tail replenishment fragment did not change significantly with the control.Real time-PCR results showed that the expression of Djpiwi-1 and Djpiwi-b was significantly down-regulated after interfering with the gene.(4)The full-length cDNA ofthe Djhsp90 b gene was 2636 bp,the non-coding region of 94 bp at the 5 'end and the 132 bp noncoding region at the 3' end,and the largest open reading frame of 2409 bp,which encodes a sequence of 802 amino acids protein.The deduced amino acid sequence of this gene contains five HSP90 family-specific tag sequences.Whole-mount in situhybridization showed thatthe gene was widely expressed in the whole tissue of the larvae and had strong hybrid signals on both sides of the body.The hybridization signal of the buds in the regenerated individuals was strong.RNA interference of the gene,the whole body of volatiles obvious shrinkage;head regeneration tail and tail regeneration head fragment regeneration rate is slow.Real time-PCR results showed that the expression of Djpiwi-1 and Djpiwi-b was significantly down-regulated after interfering with the gene.(5)The full-length cDNA of theDjhsp90 c gene was 2573 bp,which contained a maximum open reading frame of 2394 bp in length,encoding a protein encoded by a 797 bp amino acid,a noncoding region of 114 bp at the 5 'end,a 3' There are 65 bp non-coding areas.That contains six HSP90 family of genes unique to the tag sequence.Whole-mount in situ hybridization showed thatthe hybridization signal of the gene was mainly distributed on both sides and the tail of the whole body.The budding site of the regenerated fragment had strong hybridization signal.After RNA interference,the swollen phenomenon occurred on both sides of the whole volute,and the regeneration of the head and tail regeneration head was inhibited.Real time-PCR results showed that the expression of Djpiwi-1,Djpiwi-b and Djh2 b genes was significantly down-regulated after interfering with the gene.Above results showed thatDjhsp70 e,Djhsp90a,Djhsp90 b,Djhsp90b and Djhsp90 c genes had protective effects in the stress condition.The four genes were used to regulate the tissue balance of the stem cells by regulating the stem cells and played an important role in the regeneration process.In addition,the Djhsp90 c gene regulates the proliferation and differentiation of stem cells in the larvae by adjusting the Djpiwi-b and Djh2 b genes,which is an important regulatory factor in the process of regeneration.