Impact Of Sericin SER3 Expressed In Posterior Silk Gland On The Hemolymph Melanization Immunity In Transgenic Silkworm,Bombyx Mori

Using gene editing tools to modify silk fibers and using silk gland bioreactor to produce exogenous functional proteins efficiently are major subjects in the sericulture.However,a large number of attempts to directly introduce the genes expressed excellent and characteristic silk protein fibers into genome of Bombyx mori,as well as transgenic silkworms,which widely used as silk gland bioreactors,have generally encountered some bottleneck problems,such as low individual vitality,silk gland deformity,decreased reproductive capacity,and exogenous proteins inefficient expression.Therefore,it is of great significance to study the effects of exogenous gene expression on metabolism and immune system by a suitable transgenic silkworm model.In this paper,transgenic mutant silkworm pure line SER(Ser3+/+)was used for research.It's posterior silk glands could express sericin potein(SER3),which was specifically expressed in the middle silk glands.SER silkworm has avoided the problems of tissues and individual's abnormal growth and development,even the capacity of producting silk proteins was better than wild type,but hemolymph melanization process has significantly changed,which was important to innate immunity.In this paper,the regulatory mechanism of hemolymph melanization was studied in depth,and the effects of gene insertion on the melanization immunity and individual's stress resistance were discussed,we aimed to provide reference for transgenic silkworm safety assessment on the molecular level.The main results were as follows:(1)Transgenic silkworm expressed Ser3 gene in posterior silk glands,which down-regulated hemolymph melanization immunity by inhibiting the expression and activation of prophenoloxidase,as well as improving the antioxidant level of hemolymph.The hemolymph phenol oxidase(PO)activity of the 5th-instar SER larvae was significantly reduced,resulted in hemolymph melanization capacity notably decreased Firstly,investigated serine protease biochemical pathway that regulated melanin synthesis and found that,on the third day of the SER 5th-instar,the serine protease inhibitors Spn5,Spn6,and Spn32 were upregulated 4.24-fold,479.36-fold and 67.84-fold respectively in the source tissue fat body compared to the wild type,the inhibitory activity of hemolymph on protease K was significantly enhanced,which negatively regulated the active cleave of serine protease(HP21&PPAE)on PPO,the PO activity was significantly reduced.These results showed that posterior silk glands expressed Ser3 gene could inhibit PPO active cleave.Secondly,investigated the transcription level of transcription factor Bmlozenge of PPO in SER hemolymph and found that,the Bmlozenge transcription level was up-regulated by 1.37-fold and 5.13-fold respectively at 5L1d and wandering stage,but the transcription levels of PPO 1 and PPO2 were significantly lower than WT.The results indicated that posterior silk glands expressed Ser3 gene could affect PPOs transcriptional regulationFinally,investigated hemolymph antioxidant metabolism and found that,during the wandering period,the transcription levels of catalase(CAT)and two subtypes of superoxide dismutase(Cu-SOD,Mn-SOD)genes were increased by 2.47-fold,3.10-fold,and 1.20-fold respectively,CAT activity increased by 3.47-fold,which induced hemolymph total antioxidant capacity(T-AOC)and inhibit ability of anti-superoxide anion free radicals(ASAFR)increased by 1.13-fold and 1.34-fold,hydrogen peroxide content decreased by 52.4%.These results suggested that posterior silk glands expressed Ser3 gene could affect hemolymph redox metabolism,improve antioxidant capacity.(2)Transgenic silkworm expressed Ser3 gene in posterior silk glands,which induced the response of humoral immunity and changed the individual's resistance to bacteria.After 5th-instar larvae hemolymph infecting with bacteria,the overall survival rate of the SER was significantly higher than that of the wild type.When infected with Gram-negative bacterium E.coli and Gram-positive bacterium S.aureus,the wild type larvae all died,but the survival rate of the SER larvae was 16.7%and 10%,respectively.Further investigated the transcription level of SER antibacterial peptides(AMPs)related to innate immune,the transcription levels of Moricin,CecropinA,Lebocin,and Attacin were significantly increased in fat body,which was the main source of hemolymph AMPs,especially on the third day of 5th-instar when the ability of silk protein synthesis was the strongest,the AMPs expression level increased by 91.21-fold,137.86-fold,190.42-fold,and 85.77-fold respectively.The above results suggested posterior silk gland expressed Ser3 gene could up-regulate the basic response of humoral immunity.Conclusion:Transgenic silkworm's posterior silk gland expressed sericin(SER3)which specifically expressed in the middle silk gland,decreased hemolymph melanization immunity by inhibiting the melanin synthesis;induced hemolymph oxidative stress basic response;enhanced individual's resistance to bacterial infection by increasing the level of AMPs involved in humoral immunity.