The Effect Of Overexpressed Ultrabithorax Gene On The Development Of Silk Glands And Study Of Mechanism In Bombyx Mori

Bombyx mori is an important agricultural economic insect in China,mainly used for silk production,and can be later used in industries such as clothing production,medical beauty,and even tissue engineering.Its silk production relies on highly specialized silk gland tissue in the body.The protein products encoded by the Hox gene are important transcription factors involved in the formation of somite,organ development,and various life activities.Studies in Drosophila melanogaster have shown that the Hox gene plays an important regulatory role in the formation and development of salivary glands in Drosophila larvae.As the specialized salivary gland of the silkworm,there were evidence that Hox gene plays an indispensable role in the formation and development of the silk gland.However,as a member of the Hox gene family,Ultrabithorax（Ubx）gene has not been reported.In this study,BmUbx of the Bombyx mori was taken as the research object,and BmUbx was overexpressed in the silkworm through transgenic technology to study its function in the process of silk gland development and silk protein synthesis of the silkworm.The main research results are as follows+::1.Based on the previous research work on functional genes for cocoon silk traits of the research group,BmUbx in the Hox gene family of the Bombyx mori was screened out,which may be related to development of silk gland or synthesis of silk protein in the Bombyx mori.To investigate the function of BmUbx in the development of silk glands and synthesis of silk protein in Bombyx mori,we overexpressed the BmUbx gene in silkworm BmN cells at the cellular level using the p IZT-m Chery-v5-His vector.At the individual level,the coding sequence of the BmUbx gene was inserted into the silkworm genome using the transposable system vector and microinjection methods.By screening the fluorescent markers carried in the vector,the overexpression silkworm strain of BmUbx was ultimately obtained and named Ubx^OE.The insertion site validation of the Ubx^OE genome showed that the insertion site of the transposable plasmid did not affect the genome.After extracting the total RNA of BmUbx-overexpressed BmN cells and individual samples,it was found that the expression levels of BmUbx were significantly upregulated in BmN cells,middle silk gland,posterior silk glands,and so on,which indicating that we have successfully overexpressed the BmUbx gene in BmN cells and obtained transgenic silkworm strains that overexpressed the BmUbx gene.2.In this study,a BmUbx-overexpressed strain of transgenic silkworm,named Ubx^OE,was successfully constructed using the piggy Bac transposition system based on silkworm transgenic technology.To further investigate the impact of BmUbx overexpression on silkworm,we conducted a series of investigations and verifications.Through phenotypic investigation of the BmUbx-overexpressed silkworm strain,it was found that compared with the control group,the BmUbx-overexpressed silkworm strain showed a decrease in total cocoon weight,pupa weight,cocoon shell weight,and cocoon layer rate at the individual level;At the tissue and organ levels,the overexpression of BmUbx in silkworm resulted in a lighter color of the newly formed head during molting stage,a transition of the internode membrane into a transparent skin resembling that of the oil silkworm,and a decrease in the curvature and number of posterior silk glands.Testing of posterior silk gland of the BmUbx-overexpressed silkworm strain and the control strain revealed that the expression level of silk fibroin gene in the BmUbx-overexpressed silkworm strain increased at m RNA level,but did not show significant changes at protein level.Activity testing of the promoter regions of the two main silk fibroin genes revealed that BmUbx indeed enhanced the expression of downstream genes by activated the promoters of these two genes.3.In order to further find out the impact of overexpression of BmUbx gene on silk gland at the molecular level,we used transcriptome sequencing technology to compare the gene expression patterns in the posterior silk gland of Ubx^OE strain and its control strain.The analysis of transcriptome sequencing results showed that there were significant changes in genes related to ribosome and protein synthesis in Ubx^OE strain,The intersection of transcriptome data and Ch IP-seq data of Ubx locked BmMyc,an important transcription factor,which was found to be significantly inhibited in Ubx^OE strain,and its upstream gene BmSmad3 was also be inhibited in Ubx^OEstrain.Analysis of the Ch IP-seq data of Myc found that the main enrichment function of its downstream target gene was basically consistent with the enrichment function of differentially expressed genes in the transcriptome.Therefore,it is speculated that some of the phenotypic changes caused by overexpression of BmUbx are related to the inhibition of BmMyc expression.Especially in the silk gland,although the expression of silk fibroin gene is up-regulated in Ubx^OE,the inhibition of BmMyc results in the down-regulation of the expression of genes related to ribosome and protein synthesis.As a result,the silk fibroin gene has no significant change or even a decrease in the level of protein expression.