Study On The Lethal Mechanism Of The Sch Mutant Under High-temperature In The Silkworm, Bombyx Mori

Sex-linked chocolate mutant (sch) of Silkworm(Bombyx mori) is one of body color mutant, which body and head present chocolate when they just hatched but recovey normal body color after molting. It is a recessive mutant and controlled by a single gene which located at21.5locu in Z chromosome. It also has lethal traits of late embryo under high temperature. Therefore, production varieties of monoculture male silkworm was cultivated by breeders based above two characteristics of sch. Monocultural farming of male silkworm race not only can greatly reduce production costs, but also obtain better quality of silk and high-quality silk products. Previous studies show that BmTh was responsible for the body color of sch, however, the relation between BmTh gene and temperature-sensity lethal was not clear. Therefore, we carried out the following studies for the mechanism of temperature-sensitive lethal at sch mutant and obtained the corresponding results:1. The investigation of lethal trait under high-temperature during sch embryonal stageIn this study, different stages embory of Dz and sch were treated with different temperatures (25℃,30℃and35℃) and different lengths of time (1h,3h,16h,24h,36h,50h) from the fifth day to the eighth day. After treated, eggs were turned back to normal environment until hatching. Finally, we counted hatchability of every treated group. The results showed that hatchability of two species has no correlation with temperature on the fifth day of embryonic development; it has similar hatchability with ones on the fifth day of embryo before treated for36h at35℃, while the hatchability of sch began to decrease when treated for36h at35℃and significantly decreased until treated for50h, even only1.75%. However, the hatchability of Dz had no significant change under the same conditions. These results suggested that it was more sensitive to temperature on the six days of sch embryonic development and the optimum processing conditions is36-50h at35℃.2. The detection of BmTh gene and downstream production after different temperatures treatment and the phenotypic observation of BmTh enzyme inhibitor treatmentIn this study, the expression of sch mutant gene (BmTh gene) was investigated at different temperatures. When the embryos developed to the "C"-type embryo, embryos of sch and Dz were treated (25℃and35℃) for24and48hours, respectively. RT-PCR and qRT-PCR results showed that the expression of BmTh gene increased with raising temperature and prolonging the treated time in Dz embryo. The expression of BmTh gene was also significantly increased in sch embryos when the temperature was increased to35℃for24h, while it was lower than normal under the condition of the high temperature48h. In addition, dopa is the downstream products BmTh enzyme, so HPLC was carried out to detect the content of dopa on the ninth of sch and Dz embryos at different temperatures (25℃.30℃,35℃) for16h. The results showed that dopa content in two strains continuously decreased with increasing treatment temperature, but the dopa content of Dz embryo was always higher than sch at any temperature. Meanwhile, dopa content of new hatched larvae were also detected, and it found that dopa content of Dz new hatched larvae was higher than sch. BmTh activity was inhibited by artificial adding inhibitor3-IT in Dz before they begin into the first mlot, which made the head and body became white and the semilunar markings light brown compared with the normal. The results showed that survival rate of experimental group was much lower than the control group under30℃treatments and the rate of survival became lower with the concentration of inhibitor increasing.3. Transcriptome sequencing and analysis of Dz and sch embryos treated with different temperaturesBecause sch embryonic temperature sensitive lethal period was clear, Dz、 sch samples of "C"-type embryos treated with high temperature for48h were chosen for transcriptome sequencing. The quality assessment of sequencing data showed that the distribution of quality Q30were higher than82.3%; compared with silkworm genomes, the matching percent of sequence was more than65%, of which the uniquely mapped reads was more than97%; sequencing distribution of cDNA was uniform and sequencing saturation was also very well. The above results showed that sequencing quality was quite good for next analysis, which hinted that the subsequent transcriptome analysis results were reliable. We analysed the differentially expressed genes of the same species at different temperatures and the different species at same temperatures using GO and COG classification, these data provided a lot of basic data and information for our in-depth analysis and laid a solid foundation for the latter. Compared with group of Dz-25℃or sch-25℃,318differentially expressed genes were detected in both Dz and sch strain treated with high temperature (35℃), and ones only in Dz and sch were224and242, respectively;274differentially expressed genes were found in sch strain at25℃and35℃compared with Dz treated under same condition, and differentially expressed genes, which were just found in sch at25℃or35℃, was313and210, respectively. In order to find lethal gene under high temperature in sch, we also compared above318differentially expressed genes with210ones and found24differentially expressed genes in both groups, of which9were cuticular proteins. Interestingly, BGIBMGA012595was down-regulated expressed in both group, so it might be related to lethal mechanism of sch mutant under high-temperature.4. Analysis of metabolic pathwaysBased on key enzymes reported for D. melanogaster, homologous genes involved in melanin synthesis pathway genes in silkworm were examined among differentially expressed genes of sch at25℃and35℃. The expression of TH、 yellow-d、GTPCH was significant different compared with Dz under normal and high temperature conditions. The first two genes were down-regulated, while GTPCH was up-regulated in sch. Meanwhile, chitinase, which is the key enzeme in chitinase degradation pathway, was increased (2.04-fold) in sch mutant treated at35℃.Under high temperature, it generated a total of74differently expressed genes of cuticular protein in Dz strain, including12up-regulated genes and62down-regulated genes; it has65differently expressed genes of cuticular protein in sch strain, including16up-regulated genes and49down-regulated genes. Among these genes, a total of19genes were annotated as larval cuticle protein genes, in which there are8(2up-regulated genes and6down-regulated genes) and11(4up-regulated genes and7down-regulated genes) differentially expressed genes in Dz and sch strains. Cuticular protein is an important part of integument in insect. It was not only covalently bonded with chitin, but also cross-linked with coloring substances. Cuticle protein can bind to melanin in the epidermal, which play an important role in hardened exoskeleton. Under high temperatures condition, the expression of a large number of larvae cuticle protein decreased, causing that the mount of cuticle proteins was insufficient. This affected the skin reinforcement and binding with coloring substances and might affect the sch normal hardened exoskeleton and the new larval mouthparts.