Study On Expression And Function Of Key Genes For Chitin Synthesis In Diaphorina Citri Kuwayama (Hemiptera: Liviidae)

Diaphorina citri Kuwayama harms Rutaceae plants and spreads the“cancer”of citrus—Huanglongbing（HLB）,seriously jeopardizing the development of the global citrus industry.At present,the control of D.citri mainly relies on chemical control.Still,long-term and large-scale spraying of pesticides will cause pesticide residues,further increase resistance,kill beneficial insects,and cause serious agricultural non-point source pollution.Therefore,it is urgent to seek new ways to control D.citri.The synthesis and degradation of insect chitin affect the molting and metamorphosis of insects and are crucial to the growth and development of insects.Chitin synthesis pathway genes are hot targets for pest control.however,the functions of key genes in the chitin synthesis pathway of D.citri are not yet clear.In this study,D.citri was the research object.Based on the transcriptome data,combined with RT-PCR and RACE technology,the chitin synthesis pathway of D.citri was cloned.The full-length sequences of the six key genes were analyzed by bioinformatics.The m RNA spatiotemporal expression profiles of the six key genes were explored by q RT-PCR technology.Dc HK and Dc UAP were silenced by RNAi technology to inhibit D.citri chitin quality synthesis and increased lethality.The main results are as follows:1.For the first time,the complete c DNA sequences of six key genes in the chitin synthesis pathway of D.citri were cloned,and the sequence characteristics were analyzed.The full-length open reading frames（ORFs）of the 6 key genes Dc HK,Dc G6PI,Dc GFAT,Dc GNAT,Dc PGM and Dc UAP are 1329bp,1683bp,2094bp,624bp,1596bp and 1437bp,encodes 442,560,697,207,531 and 478 amino acids,respectively;They all have multiple glycosylation sites,phosphorylation sites,and structural-functional domains.The proteins encoded by these six key genes do not have signal peptides and the hydrophilic proteins without a transmembrane structure.The tertiary structure of proteins has different degrees ofα-helix,β-sheet,and random coil.The results of phylogenetic tree construction showed that they were closely related to the homologous genes of Sogatella furcifera,Bemisia tabaci,Aureobasidium pullulans,Nilaparvata lugens,Rhopalosiphum maidis,and Leptinotarsa decemlineata.2.The m RNA spatiotemporal expression profiles of six key genes in the chitin synthesis pathway of D.citri were clarified.The six key genes of the chitin synthesis pathway of D.citri were differentially expressed in different developmental stages and tissues.Among them,the expression of Dc HK was highest in third instar nymphs（2.45）and Malpighian tube（3.84）,and the lowest in fifth instar nymphs（0.80）and feet（0.23）.Dc G6PI peaked in adult（2.08）and fat body（5.20）,followed by ovary（2.29）,and lowest in second instar nymph（0.53）and foot（0.23）.Dc GFAT third instar nymph（3.87）and fat body（6.19）showed the highest expression,followed by wing（3.14）,the lowest value was in the fifth instar nymph（0.70）and foot（0.9）.The relative expression of Dc GNAT was high in the first instar nymph（1.27）and adult stage（1.12）,but highest in the ovary（6.46）and lowest in fourth instar nymphs（0.80）and testis（0.57）.The relative expression of Dc PGM was highest in fourth instar nymphs（1.33）and fat body（6.92）,and the expression levels in eggs and adults were also high.The relative expression levels of third instar nymphs（0.44）and abdomen（0.46）were the lowest;Dc UAP was most abundantly expressed in third instar nymphs（1.93）and fat body（138.4）,while those in adults（0.98）and heads（1.00）at the lowest value.3.The first study on the effects of Dc HK and Dc UAP on chitin synthesis,growth and development of D.citriIt was clear that 600 ng/μL ds Dc HK and 800 ng/μL ds Dc UAP had the best silencing effect in the fifth instar nymphs of D.citri.After feeding ds RNA for 72 h,the weight loss in the ds Dc HK group was significantly higher than that in the ds GFP group,but there was no significant difference between the ds Dc UAP group with the ds GFP group.They were 21.40%and 21.10%in ds Dc HK and ds Dc UAP groups,respectively,which were higher than the control group.In addition,in the RNAi group,the wings of the adults were folded or twisted and could not spread normally,further decreasing the body size and shortening the wing length,showing a significant effect of RNAi interference.After RNAi,the expression levels of the other key genes in the chitin synthesis pathway were significantly down-regulated.Compared with the control group,the m RNA expressions of the other key genes were down-regulated to varying degrees after feeding ds RNA for 24-72 h.After feeding ds Dc HK,the relative expressions of Dc G6PI,Dc UAP and Dc CHS were significantly down-regulated at 24 h,while Dc Tre and Dc GFAT were significantly down-regulated at 36 h.Dc PGM and Dc GNAT were significantly down-regulated at 48 h and 60 h,respectively.For ds Dc UAP treatment,the relative expression of Dc Tre began to be significantly down-regulated at 36h,and the expressions of Dc HK,Dc G6PI and Dc GFAT were significantly down-regulated at36,48,and 24h,respectively.Dc GNAT,Dc PGM and Dc CHS all showed different expressions at 60h and later.The combined treatment of ds RNA and imidacloprid had a significant effect on the fifth instar nymphs of D.citri.For different sub-lethal concentrations of imidacloprid+RNAi treatment,the mortality of citrus psyllid was significantly increased.The mortality of citrus psyllid in LC₅₀+ds Dc HK treatment group was47.80%higher than that in ds Dc HK treatment group,and the mortality in LC₅₀+ds Dc UAP treatment group was 50.30%higher than that in ds Dc UAP treatment group.There was no significant difference in body weight loss between the imidacloprid+ds Dc HK treatment group and the ds Dc HK treatment group,but the LC50+ds Dc UAP group increased 1-fold compared with the ds Dc UAP group.There was no significant difference in the transcript level between imidacloprid+ds Dc HK treatment and ds Dc HK treatment group.On the contrary,LC50+ds Dc UAP decreased the transcription level of Dc UAP by 71.10%.Except for the target gene expression,the chitin content in the imidacloprid+ds Dc HK treatment group decreased significantly,while the imidacloprid+ds Dc UAP treatment not only significantly reduced the chitin content,but also increased with the increase of the concentration of the drug.In conclusion,the key genes for chitin synthesis in D.citri are expressed in different developmental stages and in different tissues.RNAi knockdown of Dc HK and Dc UAP genes will inhibit chitin synthesis in D.citri,increased fatality rate and lead to deformed phenotypes.ds RNA combined with imidacloprid treatment has a significant synergistic effect,indicating that HK and UAP are potential specific target genes and can be used to develop a new control strategy for using RNAi-mediated control of D.citri to control the spread of Huanglongbing.