Identification And Functional Analysis Of Heat Shock Protein Family Genes In The Brown Planthopper,Nilaparvata Lugens

Insects are small ectotherms,and temperature stress can affect the survival,population dynamics and distribution of insects.One of the strategies to adapt to the changing temperature is regulating the synthesis of heat shock proteins(HSPs),which can respond promptly to temperature stresses.Besides coping with environmental stresses,HSPs as protein chaperones also play important roles in maintaining cellular and protein homeostasis,which enhances the basic development and survival of insects under normal growth conditions.So far,the research of HSPs in insects mainly focuses on HSP70 and HSP90 families,while the studies of other families are very limited.The brown planthopper,Nilaparvata lugens(St(?)l)is one of the most destructive rice pests in Asian.Considering that the outbreak,growth and development of N.lugens can be easily affected by temperature,studies on HSPs of N.lugens are of practical importance in both theory and applications.In this study,we identified the HSP genes based on bioinformatics analysis,and provided a large-scale comprehensive analysis of the gene functions.The main results are as follows:1.This study revealed the kinds and amounts of HSPs in brown planthopper.Based on bioinformatics analysis,a total of 62 HSP family genes were identified,which belong to six families,i.e.,seven genes in s HSP family,31 genes in DNAJ,10 genes in Chaperonin(including eight CCTs,one HSP10 and one HSP60),nine genes in HSP10,three genes in HSP90,and two genes in HSP100.HSP100 family was first identified in insects.Through sequence and phylogenetic analysis,it was found that the DNAJ family exhibited a high diversity,while other HSPs were relatively conserved in evolution.2.The temporal and spatial expression patterns of HSP genes in BPH under normal physiological condition and the expression characteristics under temperature stress were analyzed.q RT-PCR results revealed that most of the HSP genes are extensively expressed across all the developmental stage.The majority of the HSPs have high expression levels in the reproductive system(ovary and testis),though there are also some genes that have a high diversity of spatio-expression patterns,indicating the potential function of HSPs in reproduction.Analysis of the expression patterns under temperature stress showed that a total of 20 HSP genes(six s HSPs,three DNAJs,nine HSP70 s and two HSP90,account for 32.3%)could be up-regulated at different developmental stages,especially at egg stage and/or adult stage.Surprisingly,we did not find any HSPs that were significantly affected by low temperature stress.3.This study revealed the important roles of 30 HSPs for normal growth,development and reproduction in BPH.We systematically performed RNAi experiments on nymphs and newly emerged females of BPH,and identified 30 HSP genes that are indispensable for this insect.Specifically,Nlshsp21.1 plays an important role in the development of eggs.Ten Nl DNAJs and 7 Nl HSP70 s are indispensable in nymph and ovary development as well as female reproduction.Eight Nl CCTs have the similar function.Knockdown of these genes not only impacts nymph development,but also reduces female and male reproductivity.RNAi with Nl Hsp10 and Nl Hsp60 also produces the same phenotype,which involves in nymph development and female reproduction.Two HSP90 genes are crucial for nymph development,female fecundity and cuticle structure.4.This study provides clues to the interrelationship within or between HSP families.It was found that the phenotypes of Nl HSC70-3,Nl HSC70-4 and Nl HSC70-5were very similar after gene knockdown.RNAi with Nl DNAJA3,Nl DNAJC19 and Nl Hsp10/Nl Hsp60 resulted in coking or blackening phenotype at the edge of the insect body.In addition,the phenotype of silencing 8 Nl CCTs was almost consistent.Together,the results provide clues for the potential interaction or regulatory relationship between these HSP family members.5.This study confirmed the indispensable role of Nls HSP20.7 and Nl HSP68 in the tolerance of high temperature stress during embryonic development stage.Under normal condition,knockdown of these two genes didn't cause obvious phenotypes.However,the eggs laid by treated females developed abnormally and finally failed to complete the embryonic development after exposure to high temperature(37°C),indicating that these two genes played a vital role in the tolerance of heat stress during embryonic development stage.