Studies On The Genetic Basis Of Pigmented Non-Diapausing Eggs Pnd-2 In Silkworm,Bombyx Mori

Insects have developed a variety of important survival strategies to adapt to cyclical environmental changes,including dormancy,diapause and migration.Diapause is a phenomenon in which insects stop development at a certain stage of ontogeny in order to survive in the adverse environment.It is of great significance to study the genetic basis of insect diapause,which can help to understand the mechanism of insect adaptation to the environment and provide new molecular targets for insect pest control.The silkworm,a typical embryonic diapause insect,is an important model organism in the study of egg diapause.The embryonic diapause of silkworm is controlled by both maternal genetic effects and embryonic genotypes.Current studies mainly focus on the effects of maternal on the production of diapause eggs or non-diapause eggs and the substances metabolism related to diapause process,and some progress has been made.Which gene induces embryonic diapause after fertilization in silkworm?It hasn’t been reported yet.There is a pigmented and non-diapausing egg 2（pnd-2）mutant in the silkworm gene bank,the embryonic diapause or non-diapause is decided by the embryonic genotype,but not by the maternal,which provides us a good material to study the genes that influence diapause after fertilization.The pnd-2 mutant was used as research materials and the pnd-2 mutant gene was analyzed by genome sequencing data analysis.Next,the function and molecular mechanism of the gene was studied.The main results are as follows:1.Phenotypic observation of pnd-2 mutantThe eggs of pnd-2/pnd-2 and pnd-2/+^pnd-2&+^pnd-2/+^pnd-2 at 36 h,48 h and 72 h after oviposition were dissected and the embryonic development status was observed.The results showed that the eggs of pnd-2/pnd-2 were non-diapause eggs,and the eggs of pnd-2/+^pnd-2&+^pnd-2/+^pnd-2 were diapause eggs.The developmental status of diapause and non-diapause embryos began to differ at 36 hours after oviposition.The embryonic development of pnd-2/pnd-2 and pnd-2/+^pnd-2&+^pnd-2/+^pnd-2 began to stop at 48 h after oviposition.Usually,non-diapause eggs are not pigmented and always appear pale yellow.We observed the egg color formation process of pnd-2/pnd-2（non-diapaused eggs）and pnd-2/+^pnd-2&+^pnd-2/+^pnd-2（diapaused eggs）,and found that both were pigmented eggs,brown in color,but the the nondiapaused eggs showed lighter brown color compared to diapause eggs with darker brown color and the egg color difference began to appear at 36hours after oviposition.2.The candidate gene for pnd-2 was KWMTBOMO06962The pnd-2 gene was located on chromosome 12 of silkworm in classical genetic map.According to the relative position relationship of three mutants pnd-2,C and oq on chromosome 12 in the classical genetic map of silkworm,and the close linkage relationship between pnd-2 and Cameo,we targeted the screening range of pnd-2candidate genes to the upper of Cameo gene lacation.Third-generation genome sequencing of pnd-2/pnd-2 and pnd-2/+^pnd-2&+^pnd-2/+^pnd-2was performed and the sequencing data was assembled to quickly identify the pnd-2 mutant gene.Genome assembly data was used to analyze all the genes at the upper of Cameo gene location and the result showed there were 11 mutated genes were screened.Hence,multiple sequence alignment on the sequences of these sites in different strains of silkworm was performed to determine whether the mutations were unique to pnd-2mutant.Finally,we found a 2 bp deletion in the ORF frame of KWMTBOMO06962 gene in pnd-2 mutant,and this mutation was only found in pnd-2 mutant,but the function of this gene is unknown.Bioinformatics analysis showed that the deletion of 2 bp in the ORF frame of the gene caused frameshift mutation,which changed the amino acid sequence and finally led to the structural change of the protein.Therefore,this gene was selected as a candidate gene of pnd-2 and named as Bmpnd-2.The homologous genes of Bmpnd-2 were analyzed in different species,we found that these species are all diapause insects of Lepidoptera.We speculated that the Bmpnd-2 gene is unique to Lepidoptera and is involved in the diapause process of Lepidoptera.3.Bmpnd-2 gene produces the pigmented non-diapause phenotypeThe expression of Bmpnd-2 in pnd-2/pnd-2 and+^pnd-2/+^pnd-2 was analyzed by quantitative real-time PCR（qRT-PCR）.The results showed that the expression of Bmpnd-2 was not significantly different between pnd-2/pnd-2 and+^pnd-2/+^pnd-2 at 6 h,12 h and 20h after oviposition.The expression of Bmpnd-2 was significantly different in pnd-2/pnd-2 and+^pnd-2/+^pnd-2 at 24 h and 36 h after oviposition,and was abnormally increased in pnd-2/pnd-2,which is basically consistent with the different phenotypes of pnd-2/pnd-2and+^pnd-2/+^pnd-2 at 36 h after oviposition.In order to investigate whether the mutation of Bmpnd-2 causes a pigmented non-diapause phenotype,monoclonal silkworm（C-4）,diclonal silkworm（Dazao）and+^pnd-2/+^pnd-2 were used as materials which produce diapause eggs.The si RNA was injected into the newly born diapause eggs to interfere with the Bmpnd-2,and the embryonic development and egg color on the 5th day after intervention was observed.The results showed that the eggs in the control group were pigmented and the embryos entered diapause,while the embryos of the three silkworm strains began to develop forward after the Bmpnd-2 gene was interfered,and the eggs were all pigmented,but the egg color became lighter than that in the control group.Further,Bmpnd-2 gene was knockout in Dazao using CRISPR/Cas9-mediated gene editing technology and homozygous strain of Bmpnd-2 knockout was obtained.The eggs of this strain were preserved at 25℃,and the next generation still showed a pigmented non-diapause phenotype consistent with pnd-2/pnd-2,but the egg color is lighter than wild-type Dazao.This indicates that Bmpnd-2 is the determinant gene of pnd-2 mutation.4.Localization of Bmpnd-2 in cells and tissuesThe expression sites of Bmpnd-2 transcriptome in embryos at 36 h was examined by in situ hybridization.The results showed that Bmpnd-2 was expressed in all tissues of the embryo.Further,the trypsin was used to lyse Dazao 36 h embryos to obtain embryonic single cells,and the expression sites of Bmpnd-2 in embryonic cells was explored by immunofluorescence technique.The results showed that Bmpnd-2 was mainly expressed in the lysosome of cells.5.Bmpnd-2 is involved in egg color formationThe egg was remained brown but became lighter than the control group after intervention or knockout of the Bmpnd-2 gene.How does Bmpnd-2 affect the formation of egg color?Theoretically,diapause hormone（DH）can promote the synthesis of eye pigment precursor substance 3-hydroxyuranine accumulates in the ovary,and there is also a large amount of 3-hydroxyuranine in the newly laid diapause eggs,which further synthesizes eye pigment,making diapause eggs appear brown.The release of DH and the accumulation of 3-hydroxy-kynurenine in the ovaries of the mother generation（25℃,diapause induction condition）were not affected after the intervention or knockout of Bmpnd-2 gene.The eggs laid by the female moth still contained 3-hydroxy-kynurenine and would continue to synthesize eye pigment,so the eggs were still pigmented.So why are the eggs lighter?The expression level of genes related to the eye pigment biosynthesis was analyzed by qRT-PCR in the control and Bmpnd-2 interference groups.The results showed that the expression of BmKMO gene was significantly decreased.We hypothesized that Bmpnd-2 gene could promote the synthesis of 3-hydroxy-kynurenine in the silkworm eggs by promoting the expression of BmKMO gene,and participate in the egg color formation process of silkworm.6.Protein analysis of Bmpnd-2 interactionHow does Bmpnd-2 control the diapause of silkworm embryos?Bmpnd-2 is a single transmembrane protein that normally acts as a receptor.We hypothesized that Bmpnd-2binds to upstream signaling molecules and translocates signals into the cell membrane,activating the expression of some downstream genes,thus influencing diapause.Bmpnd-2-N-his and Bmpnd-2-C-flag expressing vectors were constructed from the extracellular and intracellular sequences of Bmpnd-2,respectively,and transfected into silkworm embryonic cell line BmE.The proteins interacting with Bmpnd-2-N and Bmpnd-2-C were caught by Co IP technology.The results of silver staining showed that Bmpnd-2-C had no significant difference bands,which no interaction proteins were caught,while Bmpnd-2-N had significant difference bands.GC-MS analysis for differential proteins was analyzed for differential proteins and the result shows that with Bmpnd-2-N specific binding protein include egg specific protein,casein protein kinase,heat shock protein 83,cycle dependent protein kinase（cdc2-related kinase/CDK1）.Among them,CDK1 can promote cell division from G₂ phase to M phase,and cell division in diapause eggs stops at G₂phase.We hypothesized that Bmpnd-2 may bind to CDK1 to prevent embryonic cells from G₂ phase entering M phase,thus causing diapause of silkworm eggs.In conclusion,the KWMTBOMO06962 gene was identified in this study and named as Bmpnd-2.The mutation of Bmpnd-2 can induce the pigmented non-diapause phenotype of silkworm eggs,and its mechanism was preliminarily studied.