Identification The Causal Gene And Regulatory Mechanism Of Pigmented And Non-diapausing Egg Mutant (pnd) In Silkworm,Bombyx Mori

Insects are ectotherms,whose growth and development are restricted by the surrounding environmental conditions.Extreme low temperatures,drought,and reduced food supply have severely restricted their life.Insects gradually evolved diapause characteristics in the process of continuous perception and adaptation to the surrounding environmental conditions.Diapause is a physiological phenomenon in which an organism temporarily stops its growth and development in response to an unfavorable living environment at a certain stage.Diapause can be identified by changes in external morphology,living habits and internal physiology of organisms.Bombyx mori is not only an important economic insect and a model organism of lepidoptera,but also a typical insect which entering diapause during the egg stage（embryonic phase）.The occurrence of diapause in Bombyx mori is directly related to its economic traits.At present,there are many reports on the effects of diapause hormones,temperature,light and other environmental signals on the maternal diapause of silkworm.However,little research has been done on the progeny diapause of silkworm,and the genetic basis that determines the embryonic diapause of silkworm has not been clearly analyzed.So,the study on the molecular mechanism of diapause in silkworm is of great significance for the improvement of the theory of insect diapause,the protection and utilization of economic insects,and the biological control of agricultural and forestry pests.The pigmented and non-diapausing egg mutant（pnd）in silkworm,Bombyx mori,which is a recessive mutation caused by a single gene on the 11^th chromosome of silkworm.The homozygous embryos of pnd showed pigmented and non-diapausing,while the heterozygous embryos of pnd showed pigmented and diapausing.Meanwhile,the diapause characteristics of the eggs of pnd mutants are only determined by the genotype of the progeny,and they have nothing to do with the secretion of diapause hormones and the environmental conditions perceived by the maternal.Therefore,pnd mutants as the dominant materials for identifying the genetic factors that determine the contemporary diapause of silkworm after fertilization,it provides the possibility for studying the progeny diapause of silkworm and parsing the genetic basis of embryonic diapause of silkworm.First of all,we had the physiological and biochemical characteristics of pnd mutants been analyzed,and we gained the candidate gene of pnd mutants using genome sequencing data.The gene controlled the phenotype of pnd mutants had been identified by combining with genetic molecular cloning and gene expression patterns analyzing and dual luciferase system testing.We further studied the functions of pnd gene and analyzed the molecular mechanisms of it.The results laid a foundation for studying the regulation mechanism of embryonic diapausing of silkworm.The main results of this study are as follows:1.Physiological and biochemical analysis of pnd mutantsThe pnd/pnd homozygote,pnd/+^pnd heterozygote and+^pnd/+^pndwild type eggs had been fed under 25℃,12 hours of light and 12 hours of dark at the same time.After 8days of incubation,we found that 88%of pnd/pnd homozygous eggs entered into body pigmentation phase,and 12%of it had entered into head pigmentation phase and deepened color.But the eggs of pnd/+^pnd heterozygote and+^pnd/+^pndwild type which for the same conditions of incubation as the eggs of pnd/pnd homozygote had entered into a deep diapause state.After 9 days of incubation,the eggs of pnd/pnd homozygote were found to have all hatched,while pnd/+^pnd heterozygote and+^pnd/+^pndwild type remained in a deep diapause phase.This is consistent with the previously reported phenotypes of pnd mutants.At the same time,we anatomy of the embryos of pnd/pnd homozygote,pnd/+^pnd heterozygote and+^pnd/+^pndwild type at 36h,48h and 72h,respectively.It was found that the embryos of pnd/+^pnd heterozygote and+^pnd/+^pndwild type arrested at about 48h,while the embryos of pnd/pnd homozygote developed continuously.There was no significant difference between the diapause hormone levels of pnd/pnd homozygote,pnd/+^pnd heterozygote and+^pnd/+^pndwild type females in pupal stage for three days in the blood using ELISA,which indicated that the pnd locus played a decisive role in diapause of progeny after fertilization,rather than being affected by diapause hormone releasing or not in the maternal stage.2.Genome sequencing data analysisWe used the third-generation genome sequencing technology to perform genome sequencing of the genomes of pnd/pnd homozygote,pnd/+^pnd heterozygote,respectively.Using the genome sequence data of Dazao as the reference,and the third-generation genome sequencing data were visualized and analyzed by using the genome visualization software which named the Savant Genome Browser 2.0.We had found genomic structural variation（SV）both in the gene region and the intergenic region of KWMTBOMO06872 in the 11^th chromosome segment of the genome of pnd/pnd homozygote.3.Molecular cloning and differential expression analysis of Bmtret1 geneKWMTBOMO06872 noted as Bmtret1-like in the silkworm genome database（Silk Base）,we further got sequence alignment analyzed by combining drosophila genome database（Fly Base）and NCBI found its homologous gene was trehalose transporter（Bmtret1）,so Bmtret1 was considered as the candidate gene of pnd mutants.The expression patterns of Bmtret1 in pnd/pnd homozygote,pnd/+^pnd heterozygote and+^pnd/+^pndwild type were investigated by q RT-PCR.The expression levels of Bmtret1 in pnd/+^pnd heterozygote and+^pnd/+^pndwild type were significantly higher than those in pnd/pnd homozygote at 6h,12h and 18h.At the same time,molecular cloning of Bmtret1 was performed in pnd/pnd homozygote,pnd/+^pnd heterozygote and+^pnd/+^pndwild type,respectively.It was found that the deletions of 747bp nucleotide sequence,which including 355bp deletions of the 3′UTR and 392bp deletions of the intergenic region of Bmtret1 gene were found in the genome of pnd/pnd homozygote.Furthermore,we cloned and analyzed the sequence of Bmtret1 in several silkworm strains,and 355bp deletions of the 3′UTR and 392bp deletions of the intergenic region of Bmtret1 gene were only found in the genome of pnd/pnd homozygote.The mutation site of Bmtret1 gene were verified by dual-luciferase reporter system,and found that the 355bp deletions of 3′UTR of Bmtret1 resulted in the dramatically down-regulation of m RNA level of Bmtret1.4.Bioinformatics analysis of Bmtret1 geneBioinformatics analysis of Bmtret1 gene showed that the full length of the open reading frame（ORF）of the gene was 1584bp,it encoded 527 amino acids in total.Bmtret1 gene belonged to a member of major facilitator superfamily（MFS）,and Bmtret1 protein contained 12 transmembrane domains.The synteny analysis of Tret1and genes on the both side of Bmtret1 in different lepidopterons genomes showed that the ancient ubiquitous protein1（AUP1）,mucolipin3,Tret1,TBC1 domain family14（TBC1D14）,retinol dehydrogenase11（RDH11）genes were arranged in linear order in the genome in several lepidopterons.This suggests that Bmtret1 is extremely conserved in lepidopterons.5.Studying the functions of Bmtret1 geneTo evaluate the role of Bmtret1 gene in diapause decision of silkworm embryos,we used CRISPR/Cas9-mediated genome editing to knock out Bmtret1 gene in the wild-type strain of silkworm Dazao.We synthesized two specific sg RNAs of Bmtret1by using gene transcription in vitro,and mixed with Cas9 protein,and got the mixture injected into the newly laid silkworm eggs produced by Dazao,which were fed at low temperature（15℃）.And G0 generation eggs of pnd were fed at 25℃after injection.The same mutant phenotype as pnd/pnd homozygote was obtained in the G1generation of silkworm eggs,which should have all eggs diapause,but they showed pigmented and non–diapausing.Furtheremore,we sequenced Bmtret1 gene in non-diapause individuals,and found that there were four kinds of sequence editing phenomena near the sg RNA target of this gene,all of those resulted in frameshift mutation of Bmtret1 gene to lose its functions,so as to Bmtret1 gene was knocked out successfully.These results showed that Bmtret1 gene was necessary to regulate the diapause of silkworm embryos.6.Studying the mechanism of Bmtret1 gene to regulate the formation of pnd mutantsIn order to further investigate the molecular mechanism of Bmtret1 gene to regulate pnd mutants,we detected the contents of trehalose and glycogen of progeny of pnd/pnd homozygote,pnd/+^pndheterozygote and+^pnd/+^pndwild type at 6h,12h,18h and24h,respectively.It showed that the contens of trehalose and glycogen of progeny of pnd/+^pnd heterozygote and+^pnd/+^pndwild type at 18h and 24h were significantly higher than that of pnd/pnd homozygote,it suggested that the formation of pnd mutants may be due to the differential expressions of Bmtret1 in pnd/pnd homozygote,pnd/+^pndheterozygote and+^pnd/+^pndwild type,which restricted the conversion of trehalose into glycogen.Thus,the eggs of pnd/pnd homozygote performed pigmented and non–diapausing,while the eggs of pnd/+^pnd heterozygote and+^pnd/+^pndwild type showed pigmented and diapausing.In conclusion,the mutation of Bmtret1 gene led to the phenotypes of pnd mutants,and Bmtret1 gene may play an important role in providing energy for the embryonic development of pnd mutants by specifically transporting trehalose and promoting the conversion of trehalose into glycogen.