Cloning And Functional Identification Of Lysozyme In Diamondback Moth,Plutella Xylostell(L.)

Plutella xylostella（Lepidoptera:Plutellidae）is one of the most serious pests that destroy crops worldwide.It mainly harms cruciferous crops.Due to the short generation period,strong reproductive ability,and resistance to a variety of pesticides,the control of P.xylostella is becoming increasingly severe.Biological control is the main development direction of green pest control in today’s society.Insect’s natural immune system has become an important target for biological control in the future,and lysozyme is an important effector in the insect’s natural immune system.The function helps to deeply understand the immune defense mechanism of P.xylostella,provides new ideas for the biological control of P.xylostella,and also lays the foundation for the development and application of lysozyme.In this study,the E.coli heterologous expression system was used to successfully express Pxlys and further study its biological activities and functions.The main results are as follows:1.Based on the genome information of P.xylostella,a 423 bp length of Pxlys was amplified using RACE technology（Gen Bank:MN702780）.which encodes 140 amino acids,a N-terminal signal peptide containing 19 amino acids,a mature peptide encoding121 amino acids,a relative molecular mass of 15.79 KDa and a theoretical isoelectric point of 8.91.2.Optimize the lysozyme expression system.After screening the pET series of prokaryotic expression vectors,it was found that pET32a-Pxlys and pET29a-Pxlys recombinant plasmids can be expressed in E.coli BL21（DE3）and E.coli Origami 2（DE3）,but E.coli Origami 2（DE3）was more conducive to the soluble expression of Pxlys than E.coli BL21（DE3）.At the same time,the fermentation conditions of recombinant engineering bacteria E.coli Origami 2（DE3）-pET29a-Pxlys was optimized,The bacteriostatic activity was best of Pxlys,when induced by IPTG（final concentration of 0.6 mmol/L）at 16°C,150 rpm for about 16 h.3.Bacteriostatic experiments showed that Pxlys had strong antibacterial activity against both gram-positive bacterial（C.stationis,M.luteus and S.aureus）and gram-negative bacterial（E.coli,Shigella sp.and Salmonella sp.）,however Pxlys showed more resistance to gram-positive bacteria than gram-negative bacteria strong antibacterial activity.In addition,Pxlys also showed bactericidal ability against Bt.The above results indicate that Pxlys has a broad spectrum of antimicrobial activity.4.The conditions that may affect the bacteriostatic activity of recombinant lysozyme were investigated.The tests showed that the optimal temperature of Pxlys is 45℃and the optimal pH is 6.0.It was worth noting that Pxlys is still antibacterial after being treated with 95℃water bath for 15 minutes.The activity indicates that Pxlys is a high temperature resistant protein and has good potential value for industrial and agricultural applications.In addition,tests have shown that Mn²⁺,K⁺,Zn²⁺,boric acid,and Tween 80can enhance the bacteriostatic activity of Pxlys;in contrast,Cu²⁺,Ca²⁺,Mg²⁺,citric acid,and absolute ethanol reduce the bacteriostatic activity of Pxlys,while Na⁺,Fe³⁺and Tween 20 had little effect on the antibacterial activity of Pxlys.5.Scanning electron microscope was used to study the bacteriostatic mechanism of lysozyme,and it was found that Pxlys can cause the surface of C.stationis to be rough and uneven,the cells to appear cracks,the contents leaked,and the cytoskeleton to collapse,eventually leading to cell disintegration and a large number of cell debris.However,E.coli showed that the cells swelled and bulged to cause cell swelling and cell rupture,which showed different degrees of cell rupture,suggesting that Pxlys may have different antibacterial mechanisms against gram-positive bacteria and gram-negative bacteria.In this study,The expression system was redesigned for Pxlys,screened out appropriate expression vectors and host bacteria,and optimized its fermentation conditions to promote the soluble expression of Pxlys in E.coli,and explored the broad-spectrum bacteriostatic activity and antibacterial mechanism of Pxlys,which provided new ideas and directions for the expression of antibacterial peptides in the prokaryotic system.At the same time,it provided theoretical prerequisites for the development and application of lysozyme from P.xylostella,and aslo provided a new direction for the biological control of P.xylostella.