Function Analysis Of Heat Shock Protein In Monochamus Alternatus Response To High Temperature

Due to the limited ability to regulate body temperature,heat stress can negatively restrain the growth,development and reproduction of insects.The the increase of average temperature and frequent occurrence of high temperature events caused by aggravation of global warming have posed bigger challenge to insects.In the long evolutionary process,insects have gradually formed a set of complicated behavioral,genetic,physiological and biochemical mechanisms response to heat stress.Monochamus alternatus Hope(Coleoptera: Cerambycidae),as a serious wood-boring pest,is in the meanwhile a dominant vector of Bursaphelenchus xylophilus(Aphelenchida: Parasitaphelenchidae)causing huge economic and ecological damage to forestry.M.alternatus mainly distributes in the southeast area in China,where the daily highest temperature near 37.5 °C or above is not uncommon.The thermotolerance and heat response mechanism have directly impact on the population dynamics and geographical distribution of M.alternatus,and have indirectly affected the epidemic trend of pine wood nematode disease.Heat Shock Protein(HSP)can function as molecular chaperones to protect proteins from misfolding and denaturation under heat stress.To explore the heat response mechanisms of M.alternatus adults: firstly,we examined the effect of high temperature on the survival and reproduction of M.alternatus adults to evaluate its thermotolerance;then PCR amplification,quantitative PCR,Western Blot,and immunofluorescence staining experiments were utilized to explored the expression pattern of HSPs in adults,tissues and cells;in addition,the molecular chaperone activity of the purified HSPs were examined by experiment in vitro;finally,the biological function of HSPs were confirmed by RNA interference experiments in the way of reverse genetics.The main results and conclusions were as follows:1.We examined the survival rate,feeding area,fecundity and offspring fitness of M.alternatus after exposure to high temperature to evaluate its thermotolerance.The lethal 50%time under 37.5,40,42.5 and 45? of M.alternatus males and females were observed by continuous heat stress on adults.Periodically repeated heat events of 37.5? for 3 h had no significant effect on the survival rate,feeding areas,life fecundity and offspring fitness of M.alternatus.But,high temperature of 40? and 42.5? had inhibited these parameters mentioned above.Notably,there were still some individuals surviving and reproducing when adults submitted to periodically repeated heat events of 40? and 42.5?.In addition,the adults exhibited the adjustment of oviposition behavior.In summary,M.alternatus possessed strong tolerance to high temperature.The common high temperature of 37.5? in forests have no impact on the population size of M.alternatus,but the rising frequency of 40? and above raised by global warming will cause negative effect on the population dynamics of M.alternatus.2.Thirteen MaltHSP20 s,two MaltHSP40 s and six MaltHSP70 s genes were cloned and characterized by RACE and PCR amplification.Bioinformatics analysis on these sequences showed that the sequence length,molecular weight,domain,sequence signature,secondary and three-dimensional structure were consistent with HSP20,HSP40 and HSP70 family.The prediction of subcellular localization indicated that HSPs of M.alternatus had widely distributed in the component of extracellular,nuclear,cytoplasm,mitochondria and endoplasmic reticulum.Phylogenetic tree based on the HSP sequence of other insects suggested that HSPs of M.alternatus had close genetic relationships with HSPs from Coleoptera species,especially those from Anoplophora glabripennis.All these results show that the structure of M.alternatus HSPs sequences are conserved.3.The relative expression level of the 21 HSPs when M.alternatus submitted to heat stress were explored by q PCR,the results showed that: under heat stress,the expression level of HSPs were upregulated and reached the peak in the treatment of 42.5? for 2 or 3 hours;the upregulated folds induced by heat stress of MaltHSP20-5,MaltHSP20-11 and MaltHSP70-2were highest;the molecular co-chaperone,MaltHSP40 s,and the cognate Heat Shock Protein 70 s,Malt HSC70 s,were less upregulated.The majority of M.alternatus HSPs had highest expression level in ovary or testis in both control and heat treatment group,followed by antenna,leg and elytra;the upregulation fold of HSPs after heat stress were highest in adult head.These findings indicate that MaltHSP20-5,MaltHSP20-11 and MaltHSP70-2 are vital genes when M.alternatus thrive in high temperature conditions,and protect the proteins involved in reproduction firstly.4.The soluble MaltHSP20-5,MaltHSP20-11 and MaltHSP70-2 protein were overexpressed and purified in vitro.Molecular chaperone activity measurement showed that the equal molecular weight MaltHSP20-5 and MaltHSP20-11 could protect MDH and CS from thermal aggregation,and the molecular activity of MaltHSP20-5 was higher than MaltHSP20-11.The ATPase activity of recombinant MaltHSP70-2 remained stable at 25 to 40?,and this recombinant protein enhanced the thermotolerance of Escherichia coli.These results show that HSP protein of M.alternatus exhibited high molecular chaperone activity.MaltHSP20-5,MaltHSP20-11 and MaltHSP70-2 can protect protein from heat aggregation and enhance the cell thermotolerance to contribute to defense against high temperature.5.The Western Blot experiment further confirmed that MaltHSP20-5,MaltHSP20-11 and MaltHSP70-2 contributed to the heat response of M.alternatus at the level of translation.The immunofluorescence staining of M.alternatus testis tubes showed that MaltHSP20-5 and MaltHSP70-2 were mainly distributed in the cytoplasm of primary spermatocyte.After heat shock,the mean gray value of MaltHSP20-5 and MaltHSP70-2 increased significantly.Meanwhile,MaltHSP20-5 were enriched in primary spermatocyte nuclear when M.alternatus submitted to heat shock.These findings certify that HSPs contribute to heat shock response and the protection of reproductive cell in M.alternatus.6.The expression level of MaltHSP20-5 and MaltHSP70-2 were inhibited by the RNA interference experiments.Injection 8ug double-strand RNA of MaltHSP20-5 and MaltHSP70-2could inhibit the target gene in three to five days.The downregulation of MaltHSP20-5 and MaltHSP70-2 caused shorter lethal median time and lower morality of adults when exposed to42.5? high temperature.Meanwhile,the expression level of other HSPs were examined by q PCR after the injection of ds RNA.The downregulation of MaltHSP20-5 and MaltHSP70-2 had an influence on the expression pattern of other HSPs.The inhibition of MaltHSP20-5 caused the downregulation of MaltHSP40-1 and MaltHSP70-2,whereas the inhibition of MaltHSP70-2caused the upregualtion of MaltHSP20-5 and MaltHSP40-1.These results not only certify the interaction relationship of HSPs in intro,bult also confirm HSPs participate the heat response of M.alternatus.