Transcriptome Analysis Of Bombyx Mori Larve Brain And The Function And Evolution Of BmSGF1

Bombyx mori is originated from Chinese wild silkworm, which process has gone through long artificial selection domestication. There are significant differences in their morphology, growth, spawning, silk production capacity and the ability to fly between the B. mori and B. mandarina. The silkworm served as a Lepidoptera model with lots of experimental advantages, such as a longer history of research, rich genetic resources, a large body size, a short lifecycle, a large offspring, ease of rearing and the completion of silkworm genome sequencing. Compared with the Diptera model insect Drosophila, silkworm body is relatively large, experimental operation is easier. The secreting cells of the central nervous system in B. mori are extremely rich, and nerve cells are relatively large. In addition, along the completion of silkworm genome sequencing, the study of silkworm neurodevelopment will promote the studies of silkworm functional genes, the abilities of silkworm disease resistance. Therefore, the silkworm is an ideal experimental material, which will provide a good model of nerve function studies. To reveal the mechanism of biological neural network and biological development research has a very important significance. In the organism, the nervous system has vital roles, which can make the body adapt to the complex and volatile external environment by adjusting the collective and control the various systems. From the simple neural network system in coelenterate to the advanced complex and accurately control system in human, it has experienced great progress and development in the structure and function of the nervous system in the process of biological evolution.The next generation sequencing technology has provide a method of the studies in digging of new genes, single nucleotide polymorphisms(SNP) and alternative splicing. Based on the technology of RNA-Seq, our study had analysis the transcriptome of the brain in day 3 fifth instar larvae in silkworm, including the annotation of gene expression, alternative splicing detection and SNP detection and so on. In this study, Bmsgf1 gene of silkworm was studied by antibody staining and RNAi technology. Meanwhile, we provide a primary investigation of the microevolution of Bmsgf1 and its co-expressed gene Bmsage between the B. mandarina and Bombyx mori. The main research results are as follows.(1) We used the RNA-Seq data of the brain in day 3 fifth instar larvae of silkworm. First, we filtered the low-quality reads and reads contain adaptors and obtained the clean reads. We have obtained a total of 27958302 read sequence and 2.8Gb total numbers of nucleotides reach. Finally, we obtained 30603 assembled Unigene. The classified statistics in brain of silkworm, there are 12219 genes coexpressed which has a higher coverage.(2) The annotation of Unigene of the sample can comment to NCBI Unigene on nonredundant database(NR), a total of 19992. To compare Unienge COG database on a total of 12891, a total of 25 different functions involved. There are 9111 unigenes(23.6%) can be noted that 62 GO functional classification and KEGG database to compare the results showed that a total of 12468 Unigene annotation to 252 pathways.(3) By comparing genetic model potential and existing gene annotation of the sample 10119 transcripts structure has been optimized. Through the distribution and the reference gene annotation collection of reads, in the reference genome to find 32467 Novel TUs. Through the analysis of alternative splicing events in silkworm brain transcriptome, we found that 5480 genes had undergone the alternative splicing events. The most event of alternative splicing is the alternative splicing of 3′.(4) The full-length sequence of Bmsgf1 was selected as the antigen for antibody preparation. pET-28a-Bmsgf1 recombinant expression plasmid was constructed and expressed in E. coli BL21(DE3). The results of SDS-PAGE indicated that the recombinant protein His-BmSGF1 was expressed in inclusion. The results of ELISA and Western blot showed that the prepared antibody displayed the antigen-specific immune responses.(5) With day 3 fifth instar larvae of silkworm, different organizations of cDNA expression analysis of Bmsgf1 was carried out. We found that Bmsgf1 expresses in the head, brain, middle silk gland, posterior silk gland, malpighian tubule and the intestinal expression, antibody staining also confirmed this. BmSGF1 and Bmsage in silkworm silk glands and nerve are co-expressed, and it also shows that there may be some regulation of the relationship between these two transcription factors.(6) We studied the function of BmSGF1 by RNAi technology. Partial sequences of Bmsgf1-1(355 bp) and Bmsgf1-2(510 bp) were selected as templates to synthesize the dsRNAs of the Bmsgf1 gene, called Bmsgf1-1 and Bmsgf1-2, respectively. To address this possibility, we used RNAi to silence the expression of Bmsgf1 in the embryonic stage. The expression of Bmsgf1 in the embryos injected with Bmsgf1-1 dsRNA was significantly reduced compared with that of embryos injected with control egfp dsRNA. In the control embryos, the nervous system was normal. In the Bmsgf1 RNAi embryos injected with Bmsgf1-1 dsRNA, development of the axon was significantly affected. By contrast, the expression of Bmsgf1 had no obvious changes by injection of the Bmsgf1-2 dsRNA. Consistently, the nervous system was normal in the Bmsgf1-2 dsRNA-injected embryos. These results demonstrate that BmSGF1 is critical for the development of the nervous system in the silkworm.(7) In the silkworm, the expression of Bmsage was reduced by knocking down Bmsgf1. These results suggest that BmSGF1 can activate the expression of Bmsage in the embryonic stage.(8) A phylogenetic tree was constructed using the SGF1 protein sequences from B. mori and other species, which belong to the Foxa subfamily. The phylogenetic tree is consistent with the species tree. BmSGF1 was closely tied with SeSGF1 of Spodoptera exigua and DpSGF1 of Danaus plexippus, but had a farther evolutionary distance with genes of other invertebrates. The homologue in Trichoplax adhaerens may be an ancestor of the Foxa subfamily.(9) We performed an alignment of BmSGF1 protein with the homologue in Homo sapiens and D. melanogaster. The DNA-binding domain(Region I) which also called the fork head domain, and the transactivation domains in the regions II and III are well highly conserved across different species. However, the transactivation domains in the regions IV and V were diverged in B. mori.(10) Based on the important position of Bmsgf1 and Bmsage, we studied the microevolution from B. mandarina to B. mori. The whole sequence of silkworm nucleotide polymorphism analysis showed that the πtotal of Bmsgf1 and Bmsage in B. mori were 0.00344 and 0.00056; 0.00718 and 0.01038 respectively in B. mandarina. Through statistical analysis, there are 52% nucleotide diversity is lost of Bmsgf1 in B. mori, and 94.6% is lost of Bmsage. The results based on θw analysis showed that the value of Bmsgf1 and Bmsage were 0.00430 and 0.00200 in B. mori and 0.00714 and 0.01142 in B. mandarina. 39.8% of the Bmsgf1 and 82.5% of Bmsage in B. mori nucleotide diversity is lost. Nucleotide polymorphisms tests, found that the silkworm Bmsage nucleotide polymorphisms are significantly lower than that of wild silkworm. It also suggests that, in the long-term evolution process, transcription factor Bmsage may by artificial selection. Of silkworm area on either side of the sage gene and gene nucleotide polymorphisms of the inspection, found that their nucleotide polymorphisms trend can present a "V" type, this also shows that significantly reduce the sage nucleotide polymorphism(SNP) is not due to the gene nucleotide polymorphism(SNP) on both sides of the area.(11) Since previous studies showed that sage plays an improtant roles during the development in silk gland, so we analyze the relative expression of Bombyx mori and B. mandarina. The results showed that compared with the B. mandarina, the expression of sage was significantly increased. This conclusion suggests that in the process of domesticated of silkworm, sage gene is likely experienced the artificial selection, and may related to the size of the cocoon.