Positional Cloning And Function Research Of L-4i(Lethal Four Instar Larvae) Gene In Silkwrom, Bombyx Mori

The silkworm is a holometabolous insect and larvae stage is important for growth and energy for entire life cycle. Therefore, there is no doubt that lethal mutant in larvae stage has enormous impact on silkworm lifecycle and caused economic loss of sericulture production. Lethal mutation in the fourth instar larvae(l-4i) was a novel found lethal mutant. In the present study, we performed positional cloning and constructing the genetic linkage map of l-4i mutant gene based on genetic analysis. Expression analysis andfunctional verification of candidate genes were performed by using q RT-PCR, RNAi, treated the wide-type silkworm by corresponding inhibitor of different concentration. The results of the study suggest that the mutation of B.mori topoisomerase gene Bmtop is mainly responsible for l-4i mutant, which laid a foundation for further study of the lethal mechanism of l-4i mutant. The progress of the study are as follows.1. Phenotypic characteristics and genetic analysis of l-4i mutantA novel lethal mutant, named lethal mutation in the fourth instar larvae(l-4i), was found during rearing of silkworm(Bombyx mori) strain P33. Compared with normal larvae, the mutant had smaller body size and slower growth rate after day 2 of the 3rd instar. The duration of the 3rd instar was extended by about 2 days. The 4th instar newly exuviated larvae barely ate mulberry leaves, almost stopped growth anddevelopment, and died successively on day 3 to day 4 in the 4th instar. Genetic analysis preliminarily indicated that the lethal mutation is controlled by a recessive gene located on euchromosome and has homogeneous lethal effect.2. Positional cloning of l-4i mutant geneAs the mutant phenotype is controlled by a recessive homogeneous gene and the individual of l-4i/l-4i is fatal based on the genetic analysis of l-4i mutant, the male parent(P1) of +/ l-4i genotype and the female parent(P2) of standard strain C108 were selected to produce the F1 offspring screened by a male cross two female. The screened F1 offsprings were used for selfing and back-crossing with P1 to product F2, BC1 F and BC1 M progeny, respectively. The polymorphic SSR markers of 28 linkage groups were selected using P1, F1 and P2. Thegenetic linkage map of the l-4i mutant gene was constructed using about 400 F2, BC1 F and BC1 M mutant individuals. The results showed that the l-4i mutant gene located on the 17 th linkage group and 13 polymorphic SSR markers linked with l-4i mutant gene, and a region of ~320 kb tightly linked to the l-4i mutant gene between the markers S2865-204 and S2865-148 was identified. 18 genes within this locus were predicted byusing gene-prediction models.3.Expression profile, gene structure and RNAi of candidate geneTo analysis the expression models of the candidate genes located on the target region, we conducted RT-PCR using the wide-type and l-4i mutant silkworm c DNA and there was no apparent differentially expressed gene. Instead, we cloned and compared the open reading frame(ORF) of 18 candidate genes between wide-type and mutant to find the mutant gene, and the result revealed that except for no difference among ORF of the most genes, only 48-bp deletion and single-base deletion occurred in the ORF sequence of B.mori topoisomerase gene(Bmtop).The result of the RNAi showed that the dsRNA-treated silkworm appeared slower growth rate, barely taken mulberry leaves, smaller body size and poor vitality as well as 24 hours later to fall into sleep compared to the control group, and subsequently begin to die, which was extremely similar to the natural mutation phenotype of l-4i mutant. Thus, the Bmtop was most likely the gene causing the l-4i mutantion.To detected the m RNA level of Bmtop at various tissues and different developmental stages, we obtained Bmtop transcript profile by q RT-PCR. The result showed that the Bmtop can be detected at almost all the tissues and developmental stages, which suggested that Bmtop is necessary for the silkworm development and also provides favorable evidence for the identification of mutant gene.4. Function validation ofl-4i mutant geneSince BMgn007081 was annotated as a topoisomerase III of silkworm in the Silkworm Genome Database, belonged to IA superfamily, to further validate the function of mutant gene, the wide-type larvae of different developmental stages were treated by Camptothecin(CPT) of 40mg/kg, 80mg/kg, 160mg/kg and then observed their morphology and growth status. The result revealed that all the wide-type silkworm treated by CPT appeared slow growth rate and smaller body size compared to the control group, and successively begin to die, which is consistent with the l-4i mutantion.The results of enzyme activity of topoisomerase between the P33 and l-4i mutant showed that the l-4i mutant have significantly reduced topoisomerase activity due to the mutation of ORF in comparison with the wide-type P33. And the trend was also detected among the larvae treated with CPT of different concentrations, furthermore, the higher concentration of CPT deal with the larvae, the lower topoisomerase activity it has. Taken together, all the findings verified that the reduced enzyme activity of topoisomerase due to deletion mutant of ORF is responsible to the l-4i mutant.The above results confirmed that the mutation of Bmtop is the main reason for the l-4i mutant, which not only provide theoretical basis for elucidation of molecular mechanisms of the l-4i mutant, but also contribute to uncover regulatory mechanism of growth and development of silkworm, moreover, possess important practical significance for precaution lethal mutation of silkworm.