Preliminary Study On Relish Participating In The Regulation Of Autophagy Of Silkworm,Bombyx Mori

The silkworm(Bombyx mori),undergoes drastic physiological changes with the degeneration and extinction of old tissues and germination of new tissues during the metamorphosis,as a completely metamorphic insect.Autophagy is one of many factors that affect tissue degradation during the metamorphic development of the silkworm.Salivary glands are known to be the main organs for most animals to secrete saliva and lubricate their mouths.This function plays an important role in assisting animals in eating.During metamorphosis,the silkworm salivary gland cells do not have mitosis,which can be used as an excellent research object.As a member of the NF-κB transcription factor family,Relish has the function of regulating the transcription of antimicrobial peptides(AMP).Recent studies in Drosophila have shown that Relish is also involved in the regulation of autophagy by up-regulating the expression of autophagy-related(Atg)Atg1.In order to study the regulatory function of Relish on autophagy during the metamorphic development of silkworm,this thesis aimed to clone the silkwormRelish genes,detect the expression of BmRelishs,Atgs,AMPs and other genes during the metamorphic development of the silkworm,and the functional analysis of the overexpression of the BmRelishs gene.To study the role of Relish in the regulation of autophagy,and to lay the foundation for exploring the interaction between innate immunity and autophagy in the developmental period.The main research results obtained in the paper are as follows:(1)According to the reported sequences of BmRelish1(NM＿001102465.1)and BmRelish2(NM＿001105234.1),the BmRelishs gene was amplified by RT-PCR from the fat body of the silkworm larvae of the Dazao variety,and two BmRelish1 sequences were obtained.The analysis of the conserved domains showed: Both sequences contain Rel homology domain(RHD)and ankyrin repeat(ANK),which belong to two subtypes of BmRelish1.The BmRelish1 with a total length of 2784 bp was named BmRelish1-a,which was as high as 97.26% identical to the reported amino acid sequence of BmRelish1;the other was 2241 bp with 78.43% identical in amino acid sequence,named BmRelish1-b.The amplified BmRelish2 sequence is consistent with the reported BmRelish2(NM＿001105234.1).(2)Detect the expression of BmRelishs in the fat body,midgut and salivary glands from the fifth instar larva of the silkworm to the pupa metamorphosis stage by real-time fluorescent quantitative PCR(Quantitative Real-time PCR,qPCR).The results show that:The expression levels of BmRelish1(a+b),BmRelish1-a and BmRelish2 in the body,midgut and salivary glands were all significantly up-regulated,especially in the salivary glands.The expression level of Relish gene in the salivary glands of the fifth instar larvae is low.The expression level of Relish1 reaches the peak at the pre-pupal and 1d of pupal stage,and then drops sharply at the 2d pupal stage;while the expression level of Relish2 increases at the 2d pupal stage.(3)qPCR detection of Atgs and AMPs in the salivary glands during the larval-pupa metamorphosis stage showed that the Atgs genes in the salivary glands increased with the developmental process during the pupal metamorphosis stage,and during the pre-pupa stage The expression of AMPs gene was up-regulated in the pre-pupa and early pupal stage.Similar to the up-regulation trend of the Relish gene in the salivary glands during the metamorphic development period,it is speculated that the up-regulation of Relish is related to the physiological processes such as immunity or autophagy during the metamorphic development period.(4)The morphological observation of the salivary glands from the fifth instar larva of the silkworm to the pupa metamorphosis development period showed that the salivary glands of the silkworm began to shrink from the pre-pupa stage,and the tissue became loose,indicating that the salivary glands at this time began to undergo tissue degradation and degradation.The results of Lysotracker Red staining of salivary glands observed with a laser confocal scanning microscope showed that the red fluorescent bright spot can be detected from 1 day in the pre-pupa period,and the red fluorescence is the strongest at 2days in the pre-pupa period,indicating that the silkworm salivary glands are from the migratory period Autophagy began to occur and reached a high level at 2 days in the pre-pupa period.(5)BmRelish1-a,BmRelish1-b,Relish2 and activated Relish1 were inserted into p IEx4 expression vector,and the recombinant expression vector was constructed and transfected into Bm N cells.Western blot analysis showed that BmRelish was successfully expressed in Bm N cells.It was found by qPCR that overexpression of BmRelish1-a and BmRelish2 increased the expression of some Atgs genes.The above research results preliminarily indicate that the silkwormRelish transcription factor has a regulatory function involved in autophagy,suggesting that the autophagy and immune pathways during the metamorphic development of the silkworm may be mutually regulated by the common regulatory factor Relish.