Fine Mapping Analysis Of The Mutant Gene In The Mini Mutant(Mini) Of Silkworm,Bombyx Mori

As a complete metamorphosis insect,the larval growth and development of silkworm（Bombyx mori）was crucial to the continuation of generations.The mini mutant was found from the wild-type strain S8V,which had the characteristics of retardation in development and homozygous lethal.In this study,based on the genetic analysis,positional cloning was used to map the mutant gene of the mini mutant,as well as to establish the genetic linkage map.Real time quantification PCR（q RT-PCR）and sequence cloning were employed for expression and structure analysis of candidate genes to conform the key gene that respect to the mini mutant.Then,combining the RNAi and RNA-seq analysis,we identified related genes participated in the formation and regulation of the mini mutant.The results of this study laid the foundation for the formation mechanism of mini mutants,and also provided the theoretical basis for the effect mechanism of the signal pathways related to the growth and development of the silkworm.The main results were listed as follows:1.Analysis of phenotypic characteristics and genetic model of the mini mutant.The size of the mini mutant was significantly smaller than that of the wild-type silkworm at the same stage.They developed very slow and successively died in the 4^thinstar,and easily distinguished from the normal silkworm in the 2^ndinstar.About 20 days after hatching,the difference of body size between the mini mutant and wild-type silkworm reached the maximum.The body weight and body length of the wild-type was about 60.13 times and3.71 times of the mini mutant respectively.The segregation ratio between wild-type and mutant type was about 3:1,indicating that the mini mutant was controlled by a heritable recessive gene and showed homozygous lethal.2.Screening and identification of differentially expressed genes in the mini mutant and wild-type（S8V）at the 48 h of 2^ndinstar.Transcriptome analysis showed a total of 2944differentially expressed genes（DEGs）,including 1638 up-regulated genes and 1306down-regulated genes.Pathway analysis showed that these genes were mainly involved in metabolic pathway and fatty acid metabolism pathway.Further analysis revealed that enzyme genes,synthesis and metabolism genes related to juvenile hormone and molting hormone also showed differences in expression levels.3.The linkage analysis and fine mapping of the mutant gene in the mini mutant.In this part,the polymorphic molecular markers on 28 chromosomes of silkworm were identified through two parents and their first offspring of hybridization,and then the mutant gene of the mini mutant was located on the 2^ndlinkage group of the silkworm employing 21 BC₁F individuals（showing phenotype of wild-type and mutant）.The candidate genes were mapped between the molecular markers nscaf 2623-SNP301 and nscaf 2623-290employing 423 BC₁M individuals（showing phenotype of mutant）,in which were5candidate genesincluding 101742339,101742200,101746751,101742051 and 101746611based on NCBI database（https://www.ncbi.nlm.nih.gov/）.4.Expression and structure analysis of candidate genes in mini mutants.The relative expression levels of 5 candidate genes were performed by q RT-PCR.Compared to the expression levels in wild-type,the relative expression levels of 101742200,101746751,101742051 and 101746611 in the 2^ndinstar were significantly different in that of the mini mutant,while the relative expression levels of 101742339,101742200 and 101742051 in the 4^thinstar were significantly different inthat of the mini mutant.The CDS sequence of101742200,101746751,101742051 and 101746611 showed no difference between the wild-type and the mutant.To our surprise,a 12-base deletion was found in the CDS of101742339 in the mini mutant,resulting in a 4-amino acids deletion in the translational level.The 101742339 gene encodes ATP-dependent metalloproteinase（YME1）,which was closely related to the energy metabolism of silkworm.Based on these facts above,we preliminarily estimated that YME1 was closely responsible to the formation of the mini mutant.5.Functional verification of YME1 using RNA interference（RNAi）.The YME1 gene was knocked down using RNA interference（RNAi）.The results showed that compared with the control group,the growth and development of silkworm were delayed and the body weight of silkworm decreased significantly in the treatment group.Meanwhile,the relative expression levels of YME1 gene and apoptosis pathway related genes（IAP1,Caspase1,Bmtrt）were also expressed significantly different.In conclusion,we preliminarily speculated that the mutation of YME1 gene may be the main reason for the formation of mini mutants,which resulted in the impairing of mitochondrial and accelerating of cell apoptosis,then resulting to abnormal physiological activities and metabolism of silkworm,and finally leading to the development retardation and successive death of the mini mutant.This study laid the foundation for the formation mechanism of mini mutants,and simultaneously provided a new target gene for the control of agricultural and forestry pests.