Research On The Regulation Of Silk Gland Development By BmFoxA In The Silkworm,Bombyx Mori

The silkworm is an important economic insect of Lepidoptera,and the silk protein it spit out in the wandering stage is processed into silk fabrics by process,which has important economic value.China is not only the origin of the silk industry,but also one of the major countries producing and exporting silk.The Silk Road not only promoted China's economic development,but also strengthened cultural exchanges between China and other countries.The silk gland is a specific organ that can secrete silk protein in the silkworm.According to its different function and structure,the silk gland can be divided into three parts: anterior,middle and posterior silk gland.The silk fibroin secreted by the posterior silk gland is enveloped by sericin secreted by the middle silk gland,and then spit out through the anterior silk gland and the silking mouth to form silk.The silk production of different types of silkworms is also different,which is related to the differences in their silk gland development.The selection and breeding of silkworms with high-yield silkworms has always been an important part of the silkworm industry.Increasing the silkworm's silkworm output is beneficial to the development of the silkworm industry.The exploration of silk gland development mechanism is helpful for us to better understand the development of silk gland,and it has certain reference significance for the transformation of high-yield silk varieties.The development of silk gland mainly includes embryonic development and postembryonic development of silk glands.The Silk gland form a fixed number of silk gland cells during the embryonic stage,and the number of silk gland cells no longer changes during the larval stage.Previous studies have shown that the protein Ras1 and the transcription factor Myc can regulate the nuclear replication of silk glands and affect the cell size.BmFoxA is a transcription factor belonging to the FoxA family cloned and found in silk glands of silkworms.Early researches found that ectopic expression of BmFoxA in silkworms can lead to ectopic development of silk gland,suggesting that BmFoxA may play a regulatory role in the development of silk gland.In addition,up-regulated expression of BmFoxA transcription factor was also detected in transgenic silkworms with Ras1 overexpression.Based on the above results,we speculate that BmFoxA may be involved in the regulation of silk gland development.We designed experiments study this hypothesis and the results are as follows:1.Acquisition and detection of transgenic individualsWe constructed a BmFoxA gene editing knock out gRNA vector,and then screened for gRNA individuals by microinjection of silkworm eggs.The silkworms were hybridized with silkworms that its Cas9 protein were expressed driven by silk fibroin gene promoter.Double positive gRNA and Cas9 silkworms were obtained by fluorescence screening.Genome testing of the double-positive silkworm found that different fragments of the BmFoxA gene were deleted.Further Western blotting was performed on the level of BmFoxA protein in the silk glands of the double-positive silkworm,and the results showed that compared with wild type,the expression of BmFoxA protein in the silk glands of the double-positive silkworm was significantly down-regulated.These experimental results indicate that we successfully obtained the transgenic individuals with posterior silk gland-specific knockout of BmFoxA,and we named it BmFox A-PSG-M.2.Effects of posterior silk gland-specific knockout of BmFoxA on silk gland developmentWe used the obtained BmFoxA-PSG-M transgenic silkworm as material,and observed the development of silk gland.We found that compared with wild type,the rear silk gland of BmFoxA-PSG-M transgenic silkworm developed abnormally,the length of the posterior silk gland was shorter and its appearance was rosary.We perform staining experiments and found that compared with wild type,the silk gland cells in the rear of BmFoxA-PSG-M silkworm were smaller,an and the silk gland cells were unevenly arranged and the size was not uniform.DAPI staining showed that the intruclear replication of the posterior silkworm nucleus of BmFoxA-PSG-M bombyx mori decreased compared with wild type.By testing the total DNA content of the posterior silk gland cells,we found that the total DNA content of the posterior silk glands of the BmFox A-PSG-M silkworm was significantly lower than that of the control group.The results showed that the transcription factor BmFoxA was involved in the regulation of posterior silk gland development.3.Effects of posterior silk gland-specific knockout BmFoxA on silk protein synthesis and secretion in silkwormThe abnormal development of the posterior silk gland of the BmFoxA-PSG-M silkworm may affect the its spinning behavior.So,we observed the silking behavior of BmFoxA-PSG-M silkworms,tested the content of silk fibroin and sericin in silk protein,and at the same time we made statistics on the economic characteristics of cocoons.Through observation,we found that part of BmFoxA-PSG-M silkworm had silking behavior,but could not spit silk normally,and finally form naked pupae.Another group of transgenic silkworms can spit thinner cocoons.The silk fibroin content in individual cocoon of BmFox A-PSG-M was extracted and calculated.The results showed that compared with wild type silkworm cocoon,the silkworm cocoon content in BmFox A-PSG-M individuals was significantly lower than 20%,and the content of sericin in cocoon was higher.We calculated the cocoon layer weight and cocoon layer rate of the silkworm.We found that compared to wild type,the cocoon layer weight of transgenic silkworms was significantly reduced.4.Effects of specific silk gland knockout of BmFoxA on silk gland developmentWe hybridized BmFoxA gRNA-positive individuals with those that its Cas9 protein were expressed driven by Ser1 gene promoter,and obtained double-positive individuals by fluorescence screening.Western blotting showed that compared with wild type,the expression of BmFoxA protein in the middle silk glands of double-positive individuals was significantly reduced which illustrate that BmFox A was successfully down-regulated in the middle silk gland.We named the obtained transgenic individuals BmFoxA-MSG-M.By microscopic observation and cell staining,we found that compared with wild type,the rear of silk glands in the middle of BmFoxA-MSG-M silkworm showed a significant shortening and thinning,and the silk gland cells also became smaller,with uneven size and disordered arrangement.5.Transcriptome analysis of the posterior silk gland of posterior silk gland-specific knockout BmFoxAWe explored the effect of BmFoxA on cell cycle and apoptosis,and found that BmFoxA can up-regulate the expression of cell cycle factors CyclinA,CyclinD and CyclinE related to DNA replication,inhibit cell apoptosis and apoptosis genes caspase1,caspase3 The expression of caspase4 and caspase9,so we speculate that BmFoxA may regulate the development of silk glands by promoting intranuclear replication on the one hand and inhibiting apoptosis on the other.To further explore the molecular mechanism,we extracted the posterior silk gland RNA of wild type silkworm and BmFox A-PSG-M transgenic silkworm on the third day of the fifth age to carry out transcriptome sequencing.The qPCR verification results indicate that the transcriptome data is reliable.We further conducted differential gene analysis on the transcriptome data,and the analysis results showed that there were a total of 1117 differentially expressed genes.There are 1010 genes up-regulated and 107 genes down-regulated compared with wild-type silkworm among them.We performed GO function enrichment analysis on the differential genes and found that GO function is mainly enriched in membrane components,cell adhesion and lipid transport,and glucose and lipid metabolism,indicating that the BmFox A gene is involved in various physiological and biochemical processes.We also carried out KEGG signal pathway enrichment analysis on differential genes and found that differential genes were mainly enriched in sugar metabolism-related pathways,such as glycolysis,pyruvate metabolism,etc.We speculate that BmFoxA may affect energy flow by participating in sugar metabolism pathways,resulting in decreased DNA replication capacity and abnormal development of silk gland cells.