HSF And HSP Gene Families In Pomacea Canaliculata:genome-Wide Identification,Evolution And Correlated Expression Pattern Under Temperature Stress

Pomacea canaliculata,as a global invasive species with extremely serious damage,had spread to 16 provinces or cities region in the southern of our country.The rapid spread of Pomacea canaliculata is closely related to its tolerance to environmental stress especially temperature,and temperature is the limiting factor for its further spread northward in China.Heat shock protein(HSP)genes are a kind of important molecular chaperone proteins which produced by organisms in response to environmental stress,and is induced to respond to the adverse effects of stimuli.Heat Shock Factor(HSF)is a key factor regulating the HSP gene.The HSF and HSP genes are ubiquitous in various organisms,and they play a crucial role in the growth and development of organisms and the resistance to abiotic stresses.Relevant studies have shown that parts of the HSP genes of P.canaliculata participates in the regulatory response to temperature stress,but there is a lack of research on the systemic family members of HSP gene family,gene family evolution,and expression regulation mode under temperature stress.In view of this,this thesis is based on the genome data of P.canaliculata and other three snails belong to the same genus or family with P.canaliculata,to identify the HSF and HSP gene sequences from the whole genome level,and to identify their gene family members,intron-exon structure,protein Bioinformatics analysis such as domain composition,chromosomal location information,and molecular phylogenetic relationship;carry out stress experiments of P.canaliculata under different temperature stresses,collect tissue samples for transcriptome sequencing,and obtain expression profile data of HSF and HSP genes in different tissues through gene expression data analysis.The expression level of the gene was verified by qPCR.The main results are as follows:1.One HSF gene was identified in the genomes of P.canaliculata and P.maculata,Lanistes nyassanus,Marisa cornuarietis,all of which were HSF1 genes.The amino acid length and gene structure of HSF1 gene in the four species were similar,but the structure of intron and exon differed greatly.Protein domain analysis showed that HSF1 genes of the four species all had the DNA-binding domains(DBD),oligomerization domains(HR-A/B),and heptavalent hydrophobic repeat region(HRC)key structures of HSF1 gene domain,and the key domain sequences are very conserved.Phylogenetic tree analysis showed that the HSF1 gene of Pomacea maculata was closest to P.maculata,followed by Marisa cornuarietis and Lanistes nyassanus,and the molecular evolutionary tree was consistent with the species evolutionary tree.Molecular evolutionary pressure analysis showed that the HSF1 gene of the ampullariidae family was evolutionarily negatively selected.Among them,the molecular evolutionary pressure ω of HSF1 gene of P.canaliculata and P.maculata is 0,indicating that the gene is subjected to strong purification selection in Pomacea genus.2.63,66,67 and 64 HSP genes were identified from the genomes of P.canaliculata,P.maculata,M.cornuarietis and L.nyassanus respectively,and 44 HSP genes in P.canaliculata were cloned and verified by molecular biology techniques.There was little difference in the total number of HSP gene family members among the four species,but there was great difference in the composition of subfamily members.Among them,HSP90 subfamily was more conserved in the four species,and the number of HSP20 subfamily genes varied greatly among different species,and the number of HSP40 subfamily genes was the largest.Phylogenetic analysis suggests the genes in HSP20 sub gene-family of P.canaliculata and P.maculata have clearly undergone expansion.Except for Chrl assembled chromosomes,there is no HSP gene distribution in the snail genome,and 58 HSP genes are distributed on the other 13 assembled chromosomes.HSP genes were mainly enriched in Chr3,Chr4,Chr7,and Chr10.The HSP20 subfamily genes were mainly distributed on Chr10 and showed tandem repeats.HSP40 genes are distributed on all chromosomes except Chrl,HSP60 genes are mainly distributed on Chr3,Chr4,Chr5,Chr8 and Chr10 chromosomes,and HSP70 genes are mainly distributed on Chr4,Chr5,Chr7 and Chr13 chromosomes.3.Nine different temperature gradients(0℃,5℃,10℃,15℃,20℃,25℃,30℃,35℃,and 40℃)and four tissue(Foot muscle,Gill,Ovary and Liver)were set and the expression profile data of HSF1 and HSP gene family genes in different tissues under different temperature stress were obtained by sample transcriptome sequencing and data analysis.The results showed that the expression levels of HSF1 gene in the four tissues from high to low were gill,foot muscle,liver and ovary.Under high temperature stress(400℃),the HSF1 gene was highly expressed in all tissues.The expression pattern of HSF1 gene in foot muscle and liver tissue was relatively consistent,but it was different in gill and ovary.It was found that 17 HSP genes were specifically highly expressed in all four tissues under high temperature stress(40℃),including 6 HSP20,3 HSP40,7 HSP70 and 1 HSP90.Then,12 genes of them were randomly selected for real-time fluorescence quantification,and the quantitative results were consistent with transcriptome analysis results.It was found that 27 HSP genes were specifically expressed in gill tissues,including 20 HSP40,6 HSP60 and 1 HSP70.Eight HSP genes were found to be specifically expressed in foot muscle tissue,including one HSP20,five HSP40 and two HSP70.This study systematically studied the composition and structural characteristics of HSF and HSP genes and gene expression patterns under different temperature stresses in P.canaliculata,providing an important theoretical basis for exploring the molecular mechanism of HSF and HSP genes and temperature tolerance.