Gene Cloning And Functional Analysis Of Two Peptidoglycan Recognition Proteins In The Diamondback Moth,plutella Xylostella(L.)

Immune system is a kind of defense system in organism to against disease.The innate immunity system of insect is simple and effective.The diamondback moth(Plutella xylostella)is one of the most destructive pest of plants.It is distributed widely and has developed strong resistance to pesticides,such as Bacillus thuringiensis,one of the biological pesticide.The reason why diamondback moth was so difficult to control was due to their powerful immune system.As a conservative pattern recognition receptor,peptidoglycan recognition proteins(PGRPs)play an important role on immune system.In this study,we have cloned 2 short PGRP genes with its whole ORF in diamondback moth P.xylostella,and we have illustrated the function and gene expression pattern in the innate immunity system.The main results were as follows:1.In accordance with the information of diamondback moth genome,RACE technology was used to clone the complete cDNA sequences of the diamondback moth gene,PGRP-S1 and PGRP-S2.Domain analysis have found both of their protein sequences contain one signal peptide,one PGRP domain and one amidase domain.The two domains of these two proteins may have the function of recognition and peptidoglycan degradation,which make them play an important role in immune response of diamondback moth.2.Quantitative real-time PCR was used to detect the expression of PGRP-S1 and PGRP-S2 in diamondback moth different tissues:head,epidermis,midgut,hemolymph and fat body.We have found that PGRP-S1 was highest expressed in the midgut,and PGRP-S2 was highest expressed in the epidermis.The results showed that the respond of PGRP-S1 to two kind of bacteria were the same.The gene expression in 3 h and 6 h were about two times of the control group.Only Staphylococcus aureus can induce upregulation of PGRP-S2.The agglutination results show that PGRP-S1 protein specifically have agglutination effect to S.aureus,and PGRP-S2 protein have agglutination effect to the two kinds of bacteria.3.Antibacterial activity of recombinant protein:compared with the control,the viability of S.aureus and E.coli were down to 1%and 32%after PGRP-S1 treatment.And the corresponding viability of E.coli was 29%after PGRP-S2 treatment.In summary,we can infer that PGRP-S1 gene can respond to the invasion of gram-positive and negative bacteria,and have certain inhibitory effect on the two types of bacteria.PGRP-S2 gene can aggregate two types of bacteria and specially kill gram-negative bacteria.The two genes both had different functions in the innate immune system.