| Long non coding RNA(lncRNA)is a kind of gene transcription product with a length of more than 200 nt,but it does not have the function of encoding protein.LncRNA not only plays an important role in epigenetic inheritance,transcriptional level and post-transcriptional level,but also plays an important role in nuclear transport of chromatin,activation and regulation of proto-oncogene,immune cell differentiation and regulation of immune system.In recent years,with the development of genome sequencing technology,lncRNA has been widely concerned and studied.Bombyx mori is a model insect of lepidoptera and an important economic insect.There are differences in the economic benefits of silk production between male and female silkworms,so the sex determination and gender difference has always been an important field of silkworm research.In 2016,researchers used high-throughput sequencing technology to find that there are six lncRNAs in the intron region of Bmdsx,the key gene downstream of the sex determination cascade regulation pathway in silkworm,one of which overlaps the fourth exon of Bmdsx.In 2019,our team cloned the lncRNA and found three different splicing forms,one of which was reverse complementary to the third intron and the fourth exon of Bmdsx,and this lncRNA participated in the selection specific splicing of Bmdsx,named Bmdsx-AS1.In this paper,the biological phenotype and function of Bmdsx-AS1 were further detected by overexpression of Bmdsx-AS1,and the regulatory mechanism of Bmdsx-AS1 was explored by studying its downstream regulatory pathway and identifying its upstream regulatory factors.The main results of this study are as follows: 1.Obtaining and phenotype analysis of Bmdsx-AS1 transgenic silkworm strainIn order to obtain more different Bmdsx-AS1 over expressed transgenic silkworm strains,to further explore the function of Bmdsx-AS1.We used the recombinant plasmid of the transgenic piggyBac [3 × p3-red,Bmdsx-AS1] which has been constructed by our team to obtain the Bmdsx-AS1 transgenic silkworm strain by injecting the silkworm eggs again through the transgenic technology,and the expression of Bmdsx-AS1 in the whole silkworm was higher than that in the wild silkworm by qPCR technology,indicating that Bmdsx-AS1 was successfully overexpressed in the transgenic silkworm.The genome of the transgenic silkworm was extracted,amplified and sequenced with specific primers.It was found that the genome of the transgenic silkworm strain overexpressing Bmdsx-AS1 was inserted into chromosome 13 and located in the intergenic region.It was found that the number of male external genitalia of transgenic silkworm increased,but the external genitalia of transgenic female silkworm had no significant difference compared with wild type silkworm.2.Analysis of the downstream regulatory pathway of Bmdsx-AS1In 2014,it was reported that the development of A8 ventral segment of silkworm was affected by EGFR signaling pathway.The male genitalia of the silkworm is located in the eighth ventral segment of the silkworm.If Bmdsx-AS1 is overexpressed,will Bmdsx-AS1 affect the EGFR signal pathway? In comparison with wild type silkworms,we found that in the male and female genitalia of transgenic silkworms that overexpressed Bmdsx-AS1,we found that the EGFR signal pathway ligand spi,the star and rho genes that transport and process spi ligands,the negative feedback related gene kekl,and the hydrolysis of activated receptors and the circulation of inactive receptors The expression levels of cbl,mop,and hrs were decreased.It is suggested that overexpression of Bmdsx-AS1 may decrease the activity of EGFR signaling pathway.The expression of Bmdsx-AS1 in silkworm was reduced by RNAi technology.The specific part of Bmdsx-AS1 sequence was selected to synthesize double stranded RNA.The double stranded Bmdsx-AS1 was injected into silkworm larvae of five instars and one day,and the total RNA was extracted and turned into cDNA 48 hours later.Compared with the control group,the expression of spi,star,rho,cbl,mop and hrs increased,indicating that the activity of EGFR signal pathway increased.Combined with the experimental results of incremental expression of Bmdsx-AS1 transgenic silkworm and interference with Bmdsx-AS1,it is concluded that Bmdsx-AS1 affects the development of external genitalia of silkworm by regulating EGFR signal pathway.3.Identification of upstream regulatory factors of Bmdsx-AS1In order to further identify the upstream regulatory factors of Bmdsx-AS1,we use GenBank database to retrieve the sequence of the promoter region of Bmdsx-AS1,and predict the functional elements on the promoter of Bmdsx-AS1.The results show that the promoter region of Bmdsx-AS1 has the cis element of Abd-B binding.It has been reported that the Hox gene BmAbd-B can affect the development of the external genitalia of silkworm.Our results show that Bmdsx-AS1 overexpression causes changes in the external genitalia of silkworm,and the promoter region of Bmdsx-AS1 also predicts the binding site of Abd-B gene of Hox gene family.Therefore,we used the double luciferase activity test to detect the promoter activity of Bmdsx-AS1.It was found that BmAbd-B decreased the activity of the full-length Bmdsx-AS1 promoter.According to the binding site of BmAbd-B binding to Bmdsx-AS1 promoter,the promoter of bmdsx-as1 was truncated in different length.Compared with the full-length promoter,the activity of the promoter increased.The results showed that BmAbd-B could regulate and control the activity of the promoter of Bmdsx-AS1.The binding site of BmAbd-B on the promoter of Bmdsx-AS1 was detected by EMSA.It was found that Bm Abd-B could specifically bind to the promoter of Bmdsx-AS1.After that,BmAbd-B was incrementally expressed in BmE cells,and the expression of Bmdsx-AS1 decreased,which further proved that BmAbd-B could regulate the expression of Bmdsx-AS1.RNAi technology was used to reduce the expression of BmAbd-B in Bombyx mori.It was found that the expression of Bmdsx-AS1 increased,while the expression of spi and rho genes related to EGFR signal pathway decreased in the male individuals of BmAbd-B interference.In females,the expression of Bmdsx-AS1 decreased,while that of spi and rho increased.It is suggested that BmAbd-B can regulate the expression of Bmdsx-AS1,and thus affect the activity of EGFR signaling pathway.In conclusion,this study further explored the function and regulatory mechanism of lncRNA Bmdsx-AS1 in silkworm.It was found that Bmdsx-AS1 overexpression or interference with Bmdsx-AS1 could affect the EGFR signal pathway activity,and that BmAbd-B of Hox gene could specifically bind to the promoter of Bmdsx-AS1 and regulate the expression of Bmdsx-AS1.Our results suggest a potential regulatory pathway,i.e.Hox gene bmabd-b regulates lncRNA Bmdsx-AS1,thus affecting the activity of downstream EGFR signaling pathway and finally the development of male genitalia in silkworm.These results provide clues and data for understanding the molecular mechanism of the development and gender differentiation of the external genitalia of silkworm. |
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