Analysis Of The Expression Characteristics And RNAi Effects Of The Four Cysteine ??protease Genes TccatB25, TccatL11, TccatL13, And TcPigk Of The Red Wheat Piranha

Cysteine proteases are proteolytic enzymes that contain cysteine active residues,which can also irreversibly inhibited by thiol reactive groups.Cysteine proteases are widely distributed in viruses,fungi,mammals.In spite of basic functions in proteolytic,it is also involved in physiological diseases,antigen presentation and signal transmission.Therefore,basing on the previous results in RNA-sequencing,we fund three potential multifunctional cathepsins genes,TccatB25,TccatL11 and TccatL13.Further,we identified this genes function in immunity,reproduction,embryo and post-embryonic larval development in Tribolium castaneum,as well as the regulation mechanisms.In addition,this study also identified PigK gene in Tribolium castaneum,which is a cysteine protease in C13 family.Additionally,the development fuction of PigK gene was identified.Further,using RNA-seq,PigK downstream signal transduction regulatory network also analysis.The results as follow:TccatB25,TccatL11 and TccatL13 genes were expressed at all developmental stages and tissue.TccatB25,TccatL11 and TccatL13 was high express in late larvae,late pupae and adults,respectively.In the meanwhile,TccatB25,TccatL11 and TccatL13 abundantly express in the head,hemolymph and gut,respectively.TccatB25,TccatL11 and TccatL13 can induced by Escherchia coli(E.coli)and Staphylococcus aureus(S.aureus)in the T.castaneum larvae,which suggesting this genes play a role in immunity.After injected ds-catB25,ds-catL11,and dscatL13,the expression levels of Rel,Jnk,Stat92 E and antimicrobial peptides were repressed.In the ds-catB25 larvae,the transcription level of Att2,Def1 and Cec1 and antimicrobial peptides were repression.While,in ds-catL11 and ds-catL13 larvae,except of Att3,the expression levels of other antimicrobial peptides were inhibited by more than 53% comparing with control.Further,the mortality after E.coli and S.aureus stimulated were performed.In the control,after E.coli and S.aureus stimulated 72 hours,the cumulative mortality is 20%.While,in the ds-catB25,dscatL11,and ds-catL13 larvae the larval mortality reached more than 40%.It suggested that TccatB25,TccatL11 and TccatL13 participate in immune function by positive regulating the expression of antimicrobial peptides through Rel,Jnk and Stat92 E.Comparing with the control adults,the expression levels of FoxO,ToR,4EBP,Pi3 k and Akt were changed in ds-catB25,ds-catL11 and ds-catL13 adults.In the meantime,ds-catB25,ds-catL11 and ds-catL13 adults had incompletely developed shape,which further reduce the daily egg production.Additionally,ds-catL11 and dscatL13 will inhibited the survival rate and the average length of first generation larvae.It suggested that TccatB25,TccatL11 and TccatL13 play a role in reproductive and development of embryonic and post-embryonic larvae through the insulin pathway.The PigK Glean number is TC009622,which gene locating on chromosome 7(LG7).The mRNA full-length was 1299 bp with 4 exons and 3 introns.The ORF(open reading frame)is 1017 bp,encoding 388 amino acids.The prediction of the structure of the TcPigK protein revealed that TcPigK has a signal peptide sequence,a C13 conservative domain and two N-glycosylation sites.Further,multiple sequence alignments revealed that the TcPigK protein share conserved cysteine and histidine active site with the mammalian and Plant Legumain.TcPigK gene stable express at eight developmental stages and can detected in all tissues.In these tissues,TcPigK highly express in fat body.Further,RNAi TcPigK in larval stage,the beetle will complete lethal in pupa stages.RNAi TcPigK in pupa,the adult beetles suffer severe reproductive defects.Furthermore,the global transcriptome profiles between RNA interference(RNAi)-treated larvae(ds-PigK)and control larvae of T.castaneum were investigated and compared by RNA-sequencing.Totally,382 differentially expressed genes(DEGs)were identified between the ds-PigK and control samples.In ds-PigK,211 DEGs were up-regulated and 171 DEGs were downregulated.These DEGs were classified into 49 GO functional groups,including such functions as the proteolysis,chitin metabolic process and defense response.In addition,These DEGs were also enrich in 7 KEGG pathway including energy metabolism,amino acid and calcium ion metabolism.It is show that TcPigK closely relating to insect epidermal chitin formation and intracellular substances and energy metabolism.