| Locust plague,flood and drought were once known as the top three deadliest natural disasters in China,often leading to a chain of social reactions,which seriously increased the cost of governing the society.The locust plague usually occurs after flood and drought,suggesting that its outbreak is closely related to environmental change and its ability to adapt to drought.The understanding of this adaptability and its mechanism will act as a scientific guidance on effective prevention and control of locust plague.The culprit of agricultural disasters in the history of China is Locusta migratoria,an agricultural pest recognized internationally,which has strong ability of migration,breeding,and adaptability to dry environment,and distribute widely on earth.Once breaking out,it poses a serious threat to agricultural production.Until recently,it is still not clear what are the key genes involved in the adaptation of L.migratoria to dry environment and what is the mechanism of these genes behind the tolerance of L.migratoria to dry environment.Therefore,taking L.migratoria as the research object,this study used bioinformatics analysis method combined with the transcriptome database of L.migratoria to identify a highly-expressed gene LmDesi(that was directly homologous to the dehydration resistance gene),induced by the desiccation in the larva stage of Drosophila melanogaster.By analyzing the gene structure,protein structure,spatiotemporal expression characteristics,and environmental sensitivity of LmDesi,the most obvious part of LmDesi induced by dry environment was determined.Using RNAi technology,the effects of LmDesi deletion on phenotype,water taxis,epidermal permeability and lipid synthesis of 5th instar locust nymphs were systematically inspected,revealing the mechanism of LmDesi’s involvement in desiccation tolerance.Finally,by analyzing the influence of LmDesi on the drying tolerance during different stage of L.migratoria,i.e.,the stage of egg-nymphs,nymphnymphs and nymph-adults,the question whether LmDesi played a conservative role in the drying tolerance of L.migratoria throughout its life cycle was answered.The contributions of this thesis are as follows:(1)Analysis of gene and protein structure,spatiotemporal expression characteristics and environmental sensitivity of LmDesi;Based on the Desi gene cDNA sequence of Drosophila,we searched the L.migratoria transcriptome database via Blast N.The cDNA fragment of LmDesi obtained was 1,000 bp,and the genomicDNA length of LmDesi was73,564 bp.The gene contains 5 exons with a total length of 783 bp encoding 260 amino acids.Through SMART online analysis and Gene Doc multi-species comparison,we figured out the LmDesi protein contained one signal peptide,one transmembrane domain,one unknown function domain,two Stats SH2 binding domains and four PDZ binding domains.Phylogenetic analysis of Desi revealed that LmDesi was the most closely related insect to Frankliniella occidentalis.RT-qPCR was used to detect the spatial and temporal expression of LmDesi in the fifth instar nymphal stage of L.migratoria.The results showed,in the N5D4 days of L.migratoria,the expression of LmDesi reached the highest level and meanwhile the expression of LmDesi was highly expressed in the epidermis,foregut and hindgut formed from the ectoderm.In addition,as changing the environmental humidity and feeding foods with different water content,we tested the expression of LmDesi in the epidermis,foregut and hindgut,which are the high-expression parts of LmDesi.We found in the case where environment has low moisture or food has low water content,the effect of high expression of LmDesi is observable in the epidermis but not in foregut and hindgut due to their relatively humid internal environment;when environment has high moisture or food has high water content,no significant difference in the expression of the gene in different parts(including epidermis)was shown.(2)Discovering the LmDesi’s involvement in drought tolerance of the fifth instar of L.migratoriaUsing RNAi technique to reduce the expression of LmDesi in the fifth instar,it was found that when the expression of LmDesi was reduced at mRNA level,the adaptability of the locusts to dry environment was weakened.Under the conditions of normal environment and wheat bran with low water content,65% of the individuals showed the death phenotype caused by the ecdysis difficulty of the old epidermis,and 30% of the individuals had no abnormal death before ecdysis,and the mortality rate reached 95%.on the other hand,under the condition of high water content in the environment or food,the decrease of Lmdesi expression could alleviate the desiccation tolerance of locust.The Y-olfactory instrument was used to detect the water tendency of the L.migratoria under different conditions.It was found that the locusts who were fed normal wheat bran after silencing the gene tended to stay in the reaction arm where located the degreasing cotton ball with high water content,suggesting that the gene could enhance the sensitivity of the group to water sources,indicating that Desi was the key gene of L.migratoria to tolerate dry environment.After LmDesi RNAi,the weight and water content of L.migratoria decreased significantly,which indicated that the water holding capacity of locust epidermis was decreased.Using Hydrophilic dye(Eosin)staining method to dip the living epidermis of locusts in each group,we observed no significant difference in the staining of locust epidermis in different treatment groups,indicating that LmDesi did not change the permeability of L.migratoria epidermis.HE staining method was used to observe the abdominal epidermis after LmDesi RNAi.The old epidermis of the LmDesi silenced group was relatively thinner before molting,whenever the water content in the food was high or low.In addition,feeding wheat bran with low water content after LmDesi silencing resulted in significant exfoliation of the old epidermis from the cell layer before molting.In conclusion,LmDesi can reduce the water holding capacity of L.migratoria by changing the thickness of its epidermis.RT qPCR was used to detect the expression of lipid synthesis and transport related genes in the epidermis of L.migratoria in different treatment groups.The expression of lipid synthesis related genes in the epidermis of L.migratoria was significantly increased,only in the group of silencing the gene and feeding wheat bran with low water content;at the same time,GCMS detection showed that the content of related CHCs increased significantly only in that group,indicating that LmDesi could cope with the dry environment by regulating the high expression of genes related to epidermal lipid synthesis,increasing the content of related CHCs in epidermis,and changing the thickness of epidermis.(3)Conservative analysis of LmDesi tolerance to dry environment at different ages of L.migratoriaTo explore whether LmDesi plays a role in the drought tolerance of L.migratoria at different developmental stages.The silencing efficiency of LmDesi gene was 79% after RNAi interference on the second day of the 2nd instar nymphs.The mortality of locusts fed with wheat bran with low water content in the relative humidity of 50% RH was significantly higher than that of the control group injected with ds GFP,indicating that LmDesi plays an important role in the desiccation tolerance of L.migratoria.RT qPCR was used to detect the expression of Lmdesi in L.migratoria.on each day of egg stage.It was found that Lmdesi was highly expressed before serosa formation.After the gene was silenced by embryonic ds RNA microinjection,more shriveled individuals appeared in the eggs cultured in extremely dry environment than in the control group,suggesting that LmDesi is also involved in the process of tolerance of the L.migratoria to dry environment in the egg stage.In conclusion,LmDesi plays an important role in the adaptation of L.migratoria eggs-nymphs,nymphs-nymphs and nymphs-adults to dry environment,indicating that the function of LmDesi gene in insect dessication adaptation is conservative. |
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