| The booklouse,Liposcelis bostrychophila Badonnel（Psocoptera:Liposcelidae）,is an important storage pest distributed worldwide.Due to the irrational use of insecticide,booklouse have developed high tolerance or resistance to various grain storage protectants,fumigants,as well as physical controls,i.e.,temperature and controlled atmosphere.Previous studies on resistance mechanism of booklouse mainly focused on biochemical level,while molecular studies on resistance mechanism were scarce.Heat Shock Protein（HSP）is a kind of specific stress protein,which was produced by organisms under extreme temperature or other stresses.According to the molecular weight,heat shock protein can be divided into five families,e.g.,HSP90,HSP70,HSP60,HSP40 and sHSP.In this thesis,the HSP genes of L.bostrychophila were identified based on the genome and transcriptome data,and the expression patterns of HSP70 and HSP90 family genes under different stresses（such as high and low temperature,insecticide induction）were systematically analyzed.Additionally,RNAi technology was used further to explain the potential biological functions of HSP70 and HSP90.The main research results of this dissertation are as follows:1 Genome-wide identification and bioinformatics analysis of HSP in L.bostrychophilaA total of 61 LbHSP genes,including LbHSP90 genes（3,similarity is 43.36%）,LbHSP70 genes（8,similarity is 33.99%）,LbHSP60 genes（10,similarity is 40.97%）,LbHSP40 genes（35,similarity is 4.46%）and sHSP genes（5,similarity is 36.45%）were identified in L.bostrychophila.Sequence analysis showed that the length of amino acid sequences encoded by LbHSPs ranged from 99 to 1,004 aa,an isoelectric point ranged from 4.79 to 9.90,and molecular weight ranged from 10.81 to 113.18 k Da.Subcellular localization analysis showed that these LbHSPs were distributed in the cytoplasm（42.62%）,nucleus（8.20%）,mitochondria（24.59%）,and endoplasmic reticulum（22.95%）.Genome structure analysis showed that the number of introns contained in different LbHSPs varied greatly（0～27）,which implied that the function of LbHSPs had potential differentiation or diversified.Different HSP families have their specific family conserved motifs.The LbHSP70s and LbHSP110s share the same conserved motifs and domains,which indicates that they may have the same functions in booklouse.Meanwhile,it further supports that HSP110s should incorporate into HSP70 family.The molecular phylogenetic tree based on amino acid sequence and contig location analysis showed that tandem repeat events occur less frequently in booklouse,and the number of sHSP was not amplified,which was the reason for explaining the small number of HSPs in this booklouse.2 The expression patterns of HSP70s and HSP90s in L.bostrychophila2.1 Differential expression analysis of transcriptome of L.bostrychophila under temperature stressUsing comparative transcriptomics technique,six transcriptome libraries of L.bostrychophila was constructed after being treated at 40℃,5℃and 27.5℃for 1 h,and the analysis of differentially expressed genes showed that 1,050 genes were significantly up-regulated and 1,404 genes were significantly down-regulated after high-temperature stress.After low-temperature stress,only 38 genes were significantly up-regulated and 59 genes were significantly down-regulated.The above results indicated that booklouse are more sensitive to high-temperature stress than low-temperature stress.After high-temperature stress,the differential genes were mainly distributed in compound synthesis,transportation,and metabolism pathways.Further analysis showed that there were 17 HSPs that were significantly up-regulated in response to temperature stress,including 5 LbHSP70 genes,4 LbHSP40 genes,4LbsHSP genes,2 LbHSP90 genes and 2 LbHSP60 genes.2.2 Expression pattern analysis of LbHSP70s and LbHSP90s in response to different developmental stagesThe mRNA transcription and expression levels of LbHSP70s and LbHSP90s in eggs,first to fourth instar nymphs and adults were detected by q PCR.The results indicated that 11 genes,LbHSP70.1-6/LbHSP110.1-2 and LbHSP90.1-3,were expressed at different developmental stages with obvious different expression patterns.In terms of LbHSP70s,the expression levels of LbHSP70.5,LbHSP70.6 and LbHSP110.2 are extremely low in the first,third and fourth instar nymphs and adults.The relative expression of LbHSP70.2,LbHSP70.4 and LbHSP110.1 was the highest in adult stage.The expression levels of LbHSP70.1 and LbHSP70.3 were relatively stable in each developmental stage.In addition,almost all LbHSP70s are relatively highly expressed in the second instar nymph stage,suggesting that they may be related to the growth and development of the second instar nymph stage of this booklouse or adapting to the external environment.Simultaneously,the differential expression of LbHSP70s in different developmental stages also suggests that it may have functional differentiation.In terms of LbHSP90s,LbHSP90.1 is significantly higher expressed in the first instar nymph when compared with other developmental stages.The expression levels of LbHSP90.2 and LbHSP90.3 were relatively stable in each development stage,which indicated that LbHSP90s might participate in the physiological activities of this booklouse in each development stage.2.3 Analysis of Expression Patterns of LbHSP70s and LbHSP90s in Response to Different Temperature TreatmentsThe response expression patterns of LbHSP70s and LbHSP90s were analyzed by treating adults of booklouse at extremely high and low temperatures（35℃,40℃,45℃,–5℃,0℃and 5℃）for 2,4,6 and 8 h,respectively.The results showed that the expressions of LbHSP70.4,LbHSP70.5 and LbHSP110.1 were significantly changed under high-temperature stress（40℃and 45℃）,and LbHSP70.5 was up-regulated significantly.After being treated at 40℃for 2 h and 4 h,its expression was up-regulated by 8,000 and 9,000 folds compared to controls（27.5℃）,respectively.The expression of other LbHSP70s had no significant changes after high-temperature stress,but only LbHSP70.3 was significantly up-regulated after 8 h treatment at 40℃.In addition,LbHSP70s were insensitive to low-temperature stress,but with the decrease of treatment temperature,the expression of LbHSP70s also had a decreasing trend.Based on the above results,we speculate that LbHSP70.5 is the key HSP gene of L.bostrychophila to cope with extremely high temperatures,and LbHSP70.5 and LbHSP110.1 also play an important role in the defense process of booklouse to cope with high temperature stress.LbHSP90s has more diverse expression patterns in response to high and low-temperature stress.For example,LbHSP90.1 is significantly up-regulated after high-temperature stress such as 40℃and 45℃,but there is a treatment time effect in response to low-temperature stress.The expression of LbHSP90.2 was significantly up-regulated under 45℃high-temperature treatment,but there was no significant change in expression compared with the control group under different high-temperature treatment conditions at other time points（except for 45℃for 8 h）.Under low-temperature stress,the expression of LbHSP90.2 was up-regulated only after 6 h and 8 h treatment at 5℃.The expression of LbHSP90.3 had no significant change under different temperature stress.Based on above data,we infer that LbHSP90.1 and LbHSP90.2 may play a certain role in the process of resistance to high and low-temperature stress,while some specific LbHSP70s is more sensitive to high-temperature stress.2.4 Effects of Insecticide Induction on Transcriptional Expression of LbHSP70s and LbHSP90sThe expression patterns of LbHSP70s and LbHSP90s are varied after different insecticide exposures,and their expression levels are also affected by the effects of treatment time and recovery time.The LbHSP70.4 was significantly up-regulated after avermectin and deltamethrin induction（0.5 h and 1 h）,and malathion and beta-cypermethrin induction for 3 h,respectively.LbHSP70.5 was significantly up-regulated by malathion,deltamethrin and beta-cypermethrin for 3 h.Other LbHSPs have dose-dependent induction of abamectin and deltamethrin,and are generally significantly expressed at 0.5 h and 1 h treatment time course;However,for the exposure of malathion and beta-cypermethrin,the expression of LbHSPs can reach the peak only after a certain accumulation of insecticide dose（after 3 h）.The expression level of LbHSPs（except LbHSP70.5）was significantly higher than that in controls after36 h recovery time after avermectin exposure.After deltamethrin induction,the expression of LbHSP70.4 and LbHSP70.5 was significantly up-regulated.However,after exposure by malathion and beta-cypermethrin,only LbHSP70.5 was significantly up-regulated.Therefore,LbHSP70.5 may be an important stress gene in the process of formation of stress resistance.3 Functional study of LbHSP70s and LbHSP90s based on RNAi technologyLbHSP70.4,LbHSP70.5,LbHSP90.1 and LbHSP90.2 can be significantly up-regulated under different stress,and then we verify their further functions using RNAi technology.The target gene ds RNA was synthesized in vitro and delivered to adults of booklouse by feeding method.After 48 h,the silencing efficiency of the target gene was detected by q PCR.The result showed that LbHSP70.4,LbHSP70.5,LbHSP90.1 and LbHSP90.2 can be effectively silenced,and the silencing efficiency is38.59%,59.24%,43.29%and 37.46%,respectively.The extremely high temperature was used to treat the adults of booklouse after silencing the above target genes,and then the mortality rate was detected.The results showed that after feeding each of dsLbHSP70.4,dsLbHSP70.5 and dsLbHSP90.2,the mortality of the booklouse after high-temperature stress（45℃）increased significantly by 1.67,2.26 and 1.91 folds,respectively.After feeding dsLbHSP90.1,the mortality of booklouse after high-temperature treatment also increased,but with no significant difference.These results indicated that LbHSP70.4,LbHSP70.5 and LbHSP90.2 were the key genes that play important roles in the process of resistance to heat stress.The bioassay of malathion and beta-cypermethrin(LC20 dose)to L.bostrychophila was also determined after knocking LbHSP70.4 and LbHSP70.5 down by RNAi.The results showed that after silencing LbHSP70.4 and LbHSP70.5,the mortality of this booklouse increased significantly under the stress of sub-lethal dose of insecticide.The mortality after beta-cypermethrin treatment was increased by 3.12 and 3.83 folds,and the mortality after malathion treatment was increased by 1.70 and 1.71 folds,respectively,compared to controls.In brief,the high-level expression of LbHSP70.5 may enhance the tolerance of L.bostrychophila to insecticides and high temperature simultaneously,and which leads to the outbreak and rapid expansion of this stored product pest. |
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