Expression And Function Research Of Long Noncoding RNA In Development Regulation Induced By Molting Hormone In Silkworm,Bombyx Mori

Long non-coding RNA(lncRNA)is a kind of non-coding RNA with transcript length of more than 200 nt.In recent year,there are many reports about the roles of lncRNA in biological growth and development,metabolic stress,autophagy and apoptosis in tumors.Although lncRNA has been used in insect research to analyze the whole genome in a variety of insects and identified the functions of lncRNA involved in insect development and metamorphosis and immune response,there are relatively few studies on how lncRNA regulates functional target genes in insect.The silkworm,Bombyx mori,is a typical model of breeding organism belonging to Lepidoptera.The metamorphosis development of Bombyx mori,at the stage of egg to pupa and pupa to adult,involves the reconstruction of a variety of tissues and multiple reproductive organs.It is a good tool and material for in-depth study of the changes,functions and regulatory mechanisms during the development of Lepidoptera induced by molting hormone.The research took the silkworm as the main research object,and ecdysone(20-hydroxy-ecdysone,20-hydroxy-ecdysone,20E)was injected into the silkworm.The fat body sequencing results obtained two lncRNAs with significantly different expressions.The biological information analysis found both may be involved in the 20 E regulation of silkworm development.This study mainly explored the regulation and mechanism of gene expression in the development of lnc585 and lnc63 regulated by 20 E in silkworm.The study of lncRNAs on the regulation of the metamorphic development induced by 20 E in the silkworm,theoretically further clarifies the molecular mechanism of insect growth and development,and in practice can also provide new target molecules for the development and comprehensive utilization of beneficial insects and the research of integrated pest control.The method to detect changes in gene expression is mainly real-time fluorescent quantitative PCR technology,which uses fluorescence in situ hybridization to perform qualitative and localization analysis of lncRNAs,and uses ds RNA interference to interfere with the expression of lncRNAs to analyze functions.The main conclusions of this study are as follows:(1)Lnc585 can participate in the metamorphic development process induced by silkworm 20 E by being the precursor of miR-281.The results of fat body detection at different time points of 20 E treatment suggest that lnc585 may be the precursor of miR-281.It is predicted that the target gene Ec RB shows the same expression trend at different time points after 20 E treatment,which is inconsistent with our expected result that miR-281 inhibits the expression of Ec RB.The expression analysis of different tissues shows that the expression of lnc585 and miR-281 are both up-regulated in Malpighian tubes.In Malpighian tubes,lnc585 can be used as a precursor of miR-281 to participate in the development process induced by 20 E.The results at different developmental stages show that the expression of lnc585 and miR-281 are not synchronized and have their own specificities.Fluorescence in situ hybridization of Bm N cells found that lnc585 is distributed in the cytoplasm and nucleus.Injection of silkworm ds RNA interfered with the expression of lnc585 in the cytoplasm.When the interference effect of lnc585 in Malpighian tubes reached 77.6%,the expression of miR-281 was also reduced by 76%,which further proved that lnc585 can be used as a precursor of miR-281.But at the same time,the expression of Ec RB was also reduced by 50%,and miR-281 may have other target genes besides Ec RB.Dslnc585 interfered with the phenotypic changes of individual silkworms,and found that the 5th instar larvae decreased their body weight in the pre-pupa stage on the 6th and 7th day.(2)Lnc63 may participate in the metamorphic development process induced by 20 E by co-expressing with the secondary receptor HR3 of the 20 E signaling pathway.The results of fat body detection at different time points in the 20 E treatment showed that the expression trends of lnc63 and HR3 at the 4 time points were basically consistent.The expression in the silk gland?Malpighian tubes?fat body?testis?ovary was consistently up-regulated,and lnc63 showed more tissue specificity than HR3.The results at different developmental stages show that the expressions of lnc63 and HR3 are basically inconsistent and have their own specificities.How the two genes regulate with each other and their regulatory mechanisms during silkworm development need tobe further detected.