| Epidermal protein is an important part of insect epidermis,which plays a key role in the formation of insect epidermal penetration resistance.Cryptolestes ferrugineus is one of the most widespread grain storage pests in the world.Its increasingly prominent problem of phosphate resistance has seriously hindered the healthy development of grain storage industry in China.However,the research on the resistance mechanism of this pest is relatively lagging behind,and the mechanism is still not clear.Based on the monitoring and screening of phosphate resistance of the main stored grain pests in China,we preliminarily constructed a relatively sensitive and resistant population of C.ferrugineus,with the resistance ratio ranging from 7.18 to 1906.92 times.Furthermore,through high-throughput expression profile sequencing,it was found that many epidermal protein genes were significantly highly expressed in resistant populations,suggesting that epidermal protein also plays an important role in the formation of phosphate resistance in C.ferrugineus,and its specific functions and regulatory mechanisms need to be further studied and explored.In this paper,the C.ferrugineus with different levels of resistance to phosphine was used as the research object.The important roles of key epidermal protein genes in the formation of phosphate resistance in C.ferrugineus were analyzed by physiological,biochemical and molecular biological methods.(1)Identification of epidermal proteins associated with phosphide resistance in C.ferrugineusThrough the analysis of expression profile data,nine epidermal protein genes were found to be highly expressed in the resistant populations.The sequence analysis showed that 7 epidermal proteins existed in the chitin binding domain,of which 5belonged to the RR2 family and 2 belonged to the RR1 family.The other two epidermal proteins are members of the CPG family.The expression patterns of 9epidermal protein genes in different resistance populations of C.ferrugineus were identified by real-time fluorescence quantitative PCR.The results showed that the expression levels of the 9 genes C1～C9 in the extremely high resistance population were significantly higher than those in the sensitive population.The expression levels in extremely high resistance populations were 17.59,12.18,5.05,12.72,17.44,75.02,9.78,2.39 and 42.01 times of those in sensitive populations,respectively.(2)Temporal and spatial expression patterns of epidermal protein genes associated with phosphide resistance in C.ferrugineusThe expressions of 9 epidermal protein genes in different instars and tissue sites of C.ferrugineus were detected by real-time quantitative PCR.The results showed that the two epidermal protein genes of RR1 subfamily were highly expressed in larval stage,but were less expressed in prepupal stage and pupal stage,and almost not expressed in adult stage.The results of expression analysis of the two genes in different tissues showed that the expression level of C7 gene in different tissues had no significant difference(P>0.05),while the expression level of C8 gene was high in wing and adipose body.The expression levels of five epidermal protein genes of RR2 subfamily all increased sharply in the pupal stage and showed great difference with other developmental stages.The expression levels of these five genes in different resistant populations showed that they were expressed in the epidermis of the head,chest,abdomen,wing and foot,and the expression levels were higher in the wing.The two epidermal protein genes of CPG family were less expressed at larval stage and prepupal stage,and the gene expression increased sharply after pupation.The expression analysis of these two genes in different tissues showed that the relative expression level of C3 gene was the lowest in the epidermis of the head,the highest in the epidermis of the abdomen,and the highest in the wing.(3)Expression pattern analysis of epidermal protein stress after phosphine stressThe expression patterns of 9 epidermal protein genes under phosphine stress were analyzed by real-time fluorescent quantitative PCR.The results showed that the effect of phosphine stress on C7 and C8 genes of RR1 subfamily was firstly increased and then decreased in susceptible populations.However,the expression of C7 gene was down-regulated in the resistant population,and the expression of C8 gene was increased first and then decreased in the resistant population.After phosphate stress,the expression levels of C1,C2,C4,C5 and C9 of the RR2 subfamily changed gently in the sensitive population,while the change range was larger in the resistant population.In resistant populations,the expression levels reached the peak after 1h of phosphate stress,and were 19.54,101.01,19.54,85.79 and 1.82 times of those in the control group after 1h of treatment,respectively.Phosphine stress can induce C3 and C6 genes of CPG family to different degrees.The expression of C3 gene in susceptible population showed no obvious rule,while the expression of C6 gene was up-regulated.The expression level of C3 gene in the resistant population was 49.07 times that in the control group after 1h treatment,the expression level of C6 gene showed a downward trend with the change of phosphine treatment time.(4)Functional validation analysis of two epidermal protein genes in RR2 subfamily of C.ferrugineusTwo epidermal protein genes,C1 and C4,from the RR2 subfamily,were selected for functional verification.The results showed that the target genes of C1 and C4 could be effectively silenced when the dsRNA of C1 and C4 genes were injected into the body of C.ferrugineu,the silencing efficiency was 56% and 60%,respectively.The target gene was silenced and the phosphine sensitivity was determined.The results showed that when the expression level of the corresponding gene in the test insect decreased,the sensitivity of the test insect to phosphine also increased.Compared with the untreated group,the mortality of test insects increased by 26.3% and 32.0%,respectively.The changes of epidermal structure before and after the silencing of two epidermal protein genes in RR2 subfamily of C.ferrugineu were observed by scanning electron microscopy(SEM).The results showed that there were significant differences in wing epidermal thickness between resistant and sensitive populations(P<0.05).In addition,the epidermal thickness of two RR2 subfamily epidermal protein genes of the resistant population decreased significantly after being silenced.In summary,this thesis focuses on epidermal protein genes related to phosphide resistance of C.ferrugineus.After identifying 9 epidermal protein genes and analyzing their temporal and spatial expression and tissue localization.It was found that these 9 genes may be related to the resistance of C.ferrugineus.Furthermore,the expression pattern of epidermal protein after phosphine stress was discussed,and the function of epidermal protein was further studied.The important role of mining RR2 subfamily in the formation of phosphate resistance of C.ferrugineus was verified by scanning electron microscopy.These results not only deepen the understanding of the resistance to phosphide in C.ferrugineus,but also some key epidermal protein genes screened may become new targets for the control of C.ferrugineus. |
