Expression And Functional Analysis Of Fatty Acid Synthase Gene Of Panonychus Citri (McGregor) (Acari:Tetranychidea)

Panonychus citri（Mc Gregor）is an important agricultural pest mite distributed worldwide,severely endangering the quality and yield of citrus and causing serious constraints to the citrus industry.At present,chemical control is still the main method for controlling P.citri,but P.citri has developed varying degrees of resistance to various types of chemical agents.Therefore,it is very important to find more healthy,safe and effective methods to replace chemical prevention and control.Insect（mite）lipids are an important component of their epidermis,and their surface lipids are their first defense barrier.The synthesis of insect（mite）lipids plays an important role in their life activities.Therefore,studying key genes in the lipid synthesis pathway is of great significance.In this study,fatty acid synthetases（FAS）genes were cloned based transcriptome data of P.citri,and bioinformatics and phylogenetic analysis were carried out using online websites and software;on this basis,RT-q PCR technology was used to detect the m RNA expression differences of Pc FAS in different mite states of P.citri;In addition,the expression of Pc FAS gene was investigated after treatment with 22.4%spirotetramat suspension;Finally,RNA interference technology was used to explore the biological function of the gene.The main research results are as follows:1.The full length c DNA sequence of the Pc FAS gene was cloned.The total length of the c DNA of the Pc FAS gene in P.citri is 7807 bp,the open reading frame（ORF）is 7260 bp,encoding 2419 amino acids.The 5’untranslated region is 300 bp,and the 3’untranslated region is 247 bp.The starting codon is ATG,the ending codon is TAA,and the tail added signal（AATAA）is located 123 bp after the ending codon.The 3’UTR contains a typical Poly A tail.The deduced protein formula is C₂₄₁₈₀H₄₀₅₄₅N₇₈₀₉O₁₀₁₁₆S₁₇₆₈,with a molecular weight of approximately 659.20 KDa and a theoretical isoelectric point p I of 4.63.Analysis of N-glycosylation sites revealed a total of 13 glycosylation potential sites.The analysis of its domain showed that the protein encoded by Pc FAS gene of P.citri contained seven domains:ketoacyl synthetase（KS）,malonyltransfer-1 B-hydroxylase dehydratase（PS-DH）,B-ketoethyl reductase（PKS-KR）,enoyl reductase（PKS-ER）,acyl carrier protein（PP binding）and thioesterase（TE）domains.The phylogenetic tree was constructed based on its amino acid sequence,and the results showed that the Pc FAS gene and the FAS of Tetranychus cinnabarinus,which is also a leaf mite,first clustered into one branch.The relative expression levels of Pc FAS gene m RNA in different mite states were detected using RT-q PCR technology.The results showed that the Pc FAS gene was expressed in all mite states,with the highest expression level in female adults,significantly higher than that of larva,male adults,and deutonymph（P<0.05）,which were 1.41,1.32,and 2.58 times their expression levels,respectively.Moreover,there was no significant difference in the relative expression levels of eggs,protonymph,and deutonymph（P>0.05）.2.The spray method was used to study the toxicity of 22.4%spirotetramat suspension（Muwangte）against P.citri.The LC value,toxicity regression equation,95%confidence interval,chi squared value,correlation coefficient,etc.were calculated using SPSS 26.0.The results showed that the toxicity regression equation was y=1.2242x+0.8754,LC₁₀was 227.780 mg/L,LC₃₀was 907.499 mg/L,and LC₅₀was 2363.922 mg/L.RT-q PCR was used to detect the relative expression level of Pc FAS gene m RNA under sublethal concentrations(LC_10,LC₃₀)of spirotetramat treatment.The results showed that compared with the control group（CK）,the relative expression level of Pc FAS gene was significantly reduced（P<0.01）after sublethal concentrations(LC₁₀,LC₃₀)of spirotetramat treatment,which was 0.71 and 0.40 times higher than the control group,respectively.3.The RNAi method was used to interfere with the female adults,T.citri,in order to explore the function of the Pc FAS gene.After 48 hours of RNAi treatment with leaf plate feeding method,the egg production,hatching rate,survival rate,and lifespan of female adults were statistically analyzed.RT-q PCR technology was used to detect the changes in the relative expression level of Pc FAS gene after 48 hours of RNAi treatment.The relative content changes of fatty acid synthase（FAS）,triglycerides（TG）,and free fatty acids（FFA）were detected using the kit method.The results showed that after silencing the Pc FAS,the egg production and survival rate of female adults were significantly reduced（P<0.05）.Although the hatching rate and lifespan of female adult mites decreased,there was no significant change compared to the control group;After silencing the Pc FAS gene for 24 hours,the relative expression level of the Pc FAS gene decreased by 35.12%,and decreased by 77.66%at 48 hours（P<0.01）;After knocking out the Pc FAS gene for 48 hours,changes in the relative content of fatty acid synthase（FAS）,triglycerides（TG）,and free fatty acids（FFA）were detected,and the results showed a significant decrease（P<0.05）.