Study On The Difference Between GFAT And PFK In Regulating Chitin And Energy Metabolism Of Nilaparvata Lugens

The growth and development of brown planthopper(Nilaparvata lugens)needs to undergo bone remodeling and energy metabolism.The main component of bone is chitin,and energy metabolism mainly includes carbohydrate metabolism,glycolytic pathway and tricarboxylic acid cycle.Glutamate: Fructose-6-phosphate transaminase(GFAT)is involved in carbohydrate metabolism and chitin metabolism,and is the first rate-limiting enzyme in the hexosamine biosynthesis pathway.Phosphofructokinase(PFK)is the rate-limiting enzyme in the glycolytic pathway.Because of its low catalytic efficiency,the rate of glycolytic is strictly dependent on the catalytic ability of PFK.In this study,we screened for potential control of the N.lugens target genes based on the effects of inhibition of GFAT and PFK gene expression levels.The work of this project was as follows:Bioinformatics analysis: Analysis of known amino acid sequences showed that the open reading frames of GFAT and PFK amino acid sequences were 2112 bp and 3378 bp,respectively.The number of amino acids translated into proteins was 703 and 1125,respectively.The predicted band sizes were 79.22 kD and 27.49 kD and the predicted protein isoelectric points were 6.31 and 4.82,respectively.RNA interference and differential gene analysis: The end of the fourth instar and the beginning of the fifth instar of N.lugens were the research object.The expressions of GFAT and PFK genes in N.lugens were inhibited by microinjection.Quantitative real-time fluorescent PCR(qRT-PCR)was used to detect the expression levels of key genes on mRNA.We performed transcriptome sequencing on RNA interference-successful brown planthopper RNA samples.The sequencing results showed that after injection of dsGFAT,1006 genes were up-regulated and 8196 genes were down-regulated.After injection of dsPFK,1498 genes were up-regulated and 2943 genes were down-regulated.There were 3141 genes regulated by both GFAT and PFK,572 up-regulated genes and 2569 down-regulated genes.According to the results of differential gene detection,we classified the Gene Ontology(GO)function and analyzed its enrichment.The results showed that the classification of GO function after interfering with GFAT gene was similar to that of interfering with PFK gene,there were 705 and 329 genes with catalytic activity,respectively;367 and 162 cells in cell components.These genes were mainly involved in metabolic processes(535 and 242 respectively).Effects of inhibition of GFAT and PFK gene expressions on physiological and biochemical aspects of N.lugens: The relative expressions of GFAT and PFK genes in developmental ages and tissues were detected by qRT-PCR.The results showed that the expression levels of GFAT and PFK genes were relatively highest in the adult stage,followed by higher expression on the third day of the fourth instar nymph or the first day of the fifth instar nymph.The expression of GFAT gene was the highest in wing,followed by epidermis,which indicated that GFAT had obvious regulating effect on chitin metabolism.The expression level of PFK gene in wing was the highest,followed by head,and stable in remaining tissues.Next,the effects of GFAT and PFK genes on chitin metabolism and energy metabolism were studied.The results showed that the expressions of TRE1-1,TRE1-2,TRE2 were significantly down-regulated,and the expressions of PPGM1,PPGM2,UGPase,GS,GP,HK,G-6-Pase and so on were also inhibited after inhibiting the expression of GFAT or PFK,respectively.Inhibition of GFAT gene expression led to a significant increase in soluble trehalase activity of N.lugens.The activity of membrane bound trehalose and the content of trehalose were decreased first and then increased,while the content of glycogen showed an opposite trend,and the content of glucose was increased slightly.When the expression of PFK gene was inhibited,membrane-bound trehalase activity was decreased significantly,trehalose content changed slightly,and glucose and glycogen content were increased significantly.These results suggested that GFAT and PFK genes can regulate trehalose metabolism in N.lugens.After 48 h of RNA interference with GFAT gene,the expressions of GNPNA,UAP,PGM1,PGM2 and CHS gene were down-regulated.The relative expression levels of GNPNA,PGM2 and UAP were significantly increased after 48 h of RNA interference with the PFK gene,and significantly decreased at 72 h.The relative expression of PGM1 gene was increased significantly after 72 h of RNA interference with the PFK gene,and the expressions of CHS1,CHS1 a and CHS1 b were also significantly up-regulated,while the expression of GFAT gene was significantly down-regulated.Moreover,after RNA interference with GFAT and PFK genes,brown planthoppers appeared phenotypes of molting abnormality and wing deformity.These results indicated that GFAT and PFK genes can effectively regulate chitin metabolism of N.lugens.Among them,the PFK gene may control the chitin metabolic pathway by regulating the upstream trehalose metabolism,which leads to difficulty in chitin synthesis and ecdysis.RNA interference with GFAT or PFK gene,HK gene expression was significantly down-regulated,and the content of ATP was eventually declined significantly.These results suggested that GFAT and PFK genes can slow down the synthesis of glucose-6-phosphate substrate by decreasing the expression of HK gene,and finally lead to the decrease the content of ATP.The relative expression levels of G6PI1,G6PI2,PFK,PK1-PK5,PK7-PK10 genes interfering with GFAT gene were down-regulated in different degrees,while the relative expression level of G6PI3 gene was significantly up-regulated,and the relative expression of PK6 gene was decreased firstly and then increased.The relative expression levels of G6PI2,G6PI3,PFK,PK2,PK4,PK7-PK9 genes were significantly down-regulated after RNA interference with PFK gene,while the relative expression of PK10 gene was up-regulated.The relative expression of G6PI1 and PK gene were decreased first and then increased,while the relative expression of PK3 and PK6 genes were increased first and then decreased.The enzymatic activities of PK and MDH were consistent with gene expression trends.These results suggested that both GFAT and PFK genes play important roles in energy metabolism.