Cloning Of HSP30 Gene From Grass Carp And Its Response To GCRV Infection

Grass carp(Ctenopharyngodon idellus)is the most productive large freshwater fish breed in China,with an output of more than 5.57 million tons in 2020.Its industry is an important part of China’s agricultural economy.However,Grass carp can easily be infected with Grass carp reovirus(GCRV)hemorrhagic disease in the breeding process,which mainly affects the first-aged fry about 10 cm,and the mortality rate is up to 90%,which seriously restricts the healthy development of Grass carp breeding industry.Practice and research show that grass carp hemorrhagic disease outbreaks are closely related to temperature,and temperature stress is easy to induce the onset of GCRV.At present,the temperature-dependent pathogenic mechanism of GCRV is still unclear,which has become one of the bottleneck factors restricting the prevention and control of grass carp hemorrhagic disease.HSP30 is a member of the small molecule HSP family of heat shock proteins(HSPs)that are found only in non-mammalian vertebrates such as fish,amphibians and reptiles,and play their chaperone role by forming oligomers with molecular masses of 100 to 800 k Da.At present,the function of HSP30 in temperature stress and viral infection is not clear.In view of this,this study cloned and identified the c DNA full-length sequence of the HSP30 gene of grass carp,which has an open reading frame(ORF)of 669 bp,encoding 222 amino acids,and the amino acid sequence homology comparison found that grass carp HSP30 has a α crystal domain of the HSP30 family at the C terminus,and the N-terminus is less conserved.Secondary structure predictions show that grass carp HSP30 consists of 8 α-helix and 8 β-folds with relatively uniform distribution.Amino acid multi-sequence alignment and phylogenetic analysis showed that grass carp HSP30 and zebrafish HSP30 had the highest similarity in amino acid sequences,clustered with the fish HSP30 family,and had low similarities with other species such as X.laevis,A.mississippiensis,Chrysemys picta bellii and bird HSP30.In order to explore the relationship between HSP30 gene and grass carp hemorrhagic disease and temperature stress,we first analyzed the relative transcription level of HSP30 among healthy grass carp tissues,and RT-PCR analysis showed that grass carp HSP30 was constitutively expressed in healthy grass carp tissues and had high expression in intestinal and gill tissues.To study the effects of temperature stress and GCRV infection on HSP30 expression,grass carp or grass carp kidney cell lines(CIK)were stressed from 18 °C culture tanks(cell culture incubators)to 28 °C culture tanks(cell culture incubators),and RT-PCR analysis showed that stress at 18 °C to 28 °C significantly inhibited the transcriptional expression of HSP30,where HSP30 expression in CIK decreased to about 18% of the control group at 12 h,in gills and intestines,Temperature stress significantly inhibited the transcriptional expression of HSP30 gene,reaching a minimum at 24 h.In addition,transcriptome and RT-PCR data analysis showed that GCRV infection with CIK or grass carp significantly inhibited the transcriptional expression of HSP30.The study found that HSP30 and HSP90 showed opposite expression patterns in response to temperature stress or GCRV infection,and temperature stress or GCRV infection significantly induced transcriptional expression of HSP90.In summary,the incidence of grass carp haemorrhagic disease caused by GCRV infection shows a typical temperature dependence,and preliminary research data show that HSP30 is significantly associated with grass carp temperature stress and GCRV infection,and HSP30 may play a pro-GCRV infection function.The follow-up work will take HSP30 or HSPs family as the starting point to systematically analyze the temperature-dependent pathogenesis mechanism of grass carp hemorrhagic disease,which has important theoretical significance and application value for the development of GCRV and other temperature-dependent aquatic animal virus disease prevention and control strategies.