Functional Study Of The CTL-S-type Lectins TcCTL5, TcCTL13 And Galectins In Trisumpia Chinensis

Model insect Tribolium castaneum is stored grain pest that seriously affects crop storage,study on its innate immunity can not only reveal the innate immune regulation mechanism of Coleoptera,but also provide a new strategy for pest control.Lectins are an important class of pattern recognition receptors for initiating innate immunity in insects,and C-type lectins(CTLs)are divided into three types:CTL-S lectins,IML lectins and CTL-X lectins.The number of CTL-S lectins is the largest in T.castaneum,most of which have not been studied.it may play an important role in the innate immunity and growth of T.castaneum.Galactoside-binding lectins(GALEs)were first discovered in mammals,but the function of insect galactoside-binding lectins is rarely studied.This paper explores the two CTL-S lectins(TcCTL5 and TcCTL13),and all the galactose lectins(TcGALE1 TcGALE2 and TcGALE3)in T.castaneum to reveal the role of the innate immune defense of invertebrate.The results are as follows:1.Structural features and evolutionary relationships of CTL-S type lectin and galactose lectin in T.castaneumThere were 10 CTL-S type lectins in the genome of T.castaneum.Among them,TcCTL5,TcCTL6 and TcCTL13 were clustered into dense subbranches and they have four motifs that are unique to these three genes,which may imply that they have functions that other CTL-S do not.Phylogenetic analysis of CTL-S lectins showed that orthologous genes for TcCTL5 and TcCTL13 were present in all the five insects we studied,but no orthologous genes for TcCTL6 were present in Acyrthosiphon pisum and pediculus humanus corporis.Genetic information analysis revealed that TcCTL3 and TcCTL4 had the same motif,but TcCTL3 had exactly twice as many motifs as TcCTL4,TcCTL3 and TcCTL4 may share a common ancestor gene.Phylogenetic analysis of insect galactose lectin indicated that these GALEs formed before species differentiation,clustering is three clusters.There are three galactose lectins in T.castaneum,one in each of these clusters.According to the number of galactosin domains,we divided these galactosins into three groups:GALE-S,GALE-D,GALE-X.2.Studies on the physiological function of CTL-S type Lectins(TcCTL5?TcCTL13)from T.castaneumThe period expression pattern showed that the expression level of TcCTL5 was the highest in the late pupa stage,and TcCTL13 was expressed at all developmental stages.Tissue expression spectrum display TcCTL5 and TcCTL13 high expressed in hemolymph,central nervous system.After interference with TcCTL5,35% of the larvae died,and the T.castaneum that eclosion into adults impaired motor ability,wing hinges were blackened and leg joints were inflexibility.After the interference of TcCTL13 in larval stage,all the insects could not pupate successfully and then died.In the late pupa stage,interference of TcCTL13,all the insects could not eclosion.Our experiment verified the diverse physiological functions of CTL-S lectin.TcCTL5 and TcCTL13 were highly expressed when stimulated by bacteria and bacterial polysaccharides.Further experiments showed that the recombinant TcCTL5 and TcCTL13 could bind bacterial polysaccharides LPS and PGN in a concentration dependent manner.In the high calcium group,TcCTL5 and TcCTL13 recombinant protein could directly bind to and agglutinate five tested bacteria.In addition,TcCTL5 interference can inhibit the expression of stat(JAK/STAT)transcription factor,and inhibiting the expression of antimicrobial peptides.TcCTL13 interference can inhibit the expression of rel(IMD)transcription factor,and seriously affecting the synthesis of antimicrobial peptides.In conclusion,TcCTL5 and TcCTL13 are involved in the innate immunity and growth and development of T.castaneum,and they can be used as important target genes to provide biological strategies for pest control.3.Study on the function of galactose lectin(TcGALE1?TcGALE2 and TcGALE3)of T.castaneumThere are three galactose lectins(TcGALE1,TcGALE2 and TcGALE3)in T.castaneum,Tissue expression spectrum showed that TcGALE1,TcGALE2 and TcGALE3 high expression in the central nervous system and hemolymph.Under bacteria and bacterial polysaccharides stimulation,TcGALE1,TcGALE2 and TcGALE3 high expression.TcGALE1,TcGALE2 and TcGALE3 recombinant protein can bind to bacterial cell wall components and can also bind directly and agglutination the five species of bacteria.these results suggest that galactose lectin TcGALE1,TcGALE2 and TcGALE3 play a role in innate immunity of T.castaneum.