Regulation Of Incrna On Silk Protein Synthesis Related Genes In Silkworm Bombyx Mori

Silkworm,an important economic animal around the world and the most important model insect of lepidoptera,has a huge value in the area of industrial and scientific research.Silk protein has good ductility and mechanical properties,it can be used as materials of artificial skin,blood vessels and bones,and can also be used as a substitute for polymer materials.Silk gland is an important organ for producing silkworm.The studies on the molecular mechanism of silk protein synthesis related genes will provide important theoretical basis for the improvement of silk.We have obtained a large number of new long non-coding RNAs by sequencing the silkworm transcriptome previously,and found that some lncRNAs have some complementary sequence with some miRNAs according to the analysis of bioinformatics,and the target gene of these miRNAs was related to the genes of regulating the synthesis of silk protein.Thus,we speculated that the lncRNA and the silk synthesis genes might be ceRNA(Competitive endogenous RNA)and compete with each other to interact with miRNAs.Besides,lncRNA might exert functions as sponges of miRNAs to regulate the synthesis of silk proteins.To verify the hypothesis above,one long non-coding RNA named lnc7107 was selected,and the complementary miRNAs of lnc7107 named miR-2739 and miR-34-5p were chosen as objects.Firstly,quantitative real-time PCR was performed to detect the expression profiles of long non-coding RNAs and miRNAs in different parts and different developmental stages of silk gland,and the results showed that lnc7107 was highly accumulated in the posterior silk gland of the 5th-instar molting stage,while the miR-2739 and miR-34-5p,which were originated from the complementary strand of lnc7107,had little expression in the posterior silk gland.The Fib-H and p25,which are the target genes of miR-2739 and miR-34-5p,were the key genes of silk fibroin synthesis,and both of them have higher expression levels in the posterior silk gland.It showed that lnc7107 might be the ceRNA of Fib-H and p25,and competitive binding to miRNA.Secondly,the in vitro interaction between the miRNA and the target gene was identified by the dual luciferase activity assay in the HEK293 cell line.It was found that miR-2739 can interact with the 3'-UTR of Fib-H.After the transfection of miRNA mimics,the luciferase activity was decreased by 51.4% to a significant level,indicating that Fib-H was the true target of miR-2739.For the further study of the mechanism of lnc7107 regulating on the silk fibroin,dsRNA was used to interfere with the expression of lnc7107 in silkworm individuals,and detected the expression level of Fib-H and miR-2739.We found that the expression of lncR7107 was down-regulated,and the expression level of Fib-H was also decreased,while the expression level of miR-2739 was increased.We also observed that two silkworm cocoons appeared lighter and brighter.It is suggested that lnc7107 might be acted as a sponge of miR-2739 and compete with Fib-H for ceRNA to compete with miR-2739,thereby regulating the synthesis of silk fibroin.We also examined the interactions of lnc18326,lnc1433,and the complementary relationship with miR-2841 and miR-11-3p,as the complementary sequences of lnc18326 and lnc1433,respectively.The gene expression profiles of the silk gland on the anterior middle,and posterior period from the day 3 fifth-instar larvae to the fifth-instar molting stage in the silkworm showed that lnc18326 and lnc1433 were higher in the middle silk gland,while miR-2841 was lower in the middle of the day 3 fifth-instar larvae in silkworm,and miR-11-3p was lower in the middle silk glands than others from the day 3 fifth-instar larvae to the day 5 fifth-instar larvae in the silkworm.Subsequently,the in vitro interaction between the two miRNAs and Ser2 was identified in the HEK293 cell line by the dual luciferase activity assay.It was found that the luciferase activity was not significantly decreased after co-transfection of the miRNA mimic,Ser2 may not be the true target of both miRNAs.The dsRNA was used to infertere with the epression of two lncRNAs,and the expression levels ofthe two miRNAs and Ser2 were detected,while no significant change was observed.No abnormality was observed on the phenotype of silkworm,which suggested that lnc18326 and lnc1433 may participate in the synthesis regulation of silk fibroin by other mechanisms and miRNAs produced on their complementary strands.This result laid a good foundation for further exploration of lncRNA regulation on the synthsis of silk fibroin.In summary,we used the silk gland of silkworm as the experimental material to conduct a preliminary study on the regulation relationship between lncRNA,miRNA and its target genes,which laid a good foundation for further exploration of lncRNA involved in the regulation of silk gland development.From the perspective of lncRNA,it provides a theoretical basis for cultivating high-yield and high-quality silk varieties.