Function Of Mutanted Gene For Contraction Mutant In Silkworm,Bombyx Mori

Insects, belong to insecta of arthropods in invertebrates, are the most prosperous of the animals. It possesses the world’s largest number of animal species, which is attributed to its amazing capacity of adaptive behaviors. Heat tolerance is an important physiological characteristic in response to the changes of external environment. It had been reported in a number of insects, such as:grasshoppers, whiteflies, beetles, moths, ants, flies and wasps. However, the molecular mechanism of heat tolerance is still not clear. In the present study, a temperature-sensitive mutant silkworm cot was used, cot, showing a recessive pattern of inheritance, is one of the behavioral mutants in the silkworm, Bombyx mori. Its phenotype, which is exhibiting strong contraction, rolling, vomiting and temporary lack of movement after manual stimulation, followed by slow recovery after stimulation, is controlled by a single gene located at locus25.0on the silkworm genetic linkage group15. We observed its behavior, determined the mutant gene and investigated the influence on other metabolic pathways by transcriptome. The main results obtained are as follows:1. Observation of cot mutantTo understand the causes of cot mutant, it was induced with mechanical and thermal stimulations. At room temperature, the individuals with different genotype (cot/cot, cot/+and+/+) on day5of5instar were repeatedly rubbed. Only larvae of the cot/cot mutant showed slight contraction when their bodies were rubbed, but became normal immediately after the finish of rubbing and did not induced cot phenotypes. Some cot mutants (cot/cot) manifested the phenotype of thoracic enlargement and shortened segment when treated at30℃for5min, and all exhibited strong contraction, rolling, vomiting and a momentary cessation of movement after manual stimulation when treated for5min. The cot/+larvae showed slight contraction under the condition of45℃for10min. The wild-type Dazao (+/+) did not show any contraction even under52℃for10min. These observations showed that the sensitive of cot/cot and cotl+genotype is distinct to different temperatures. Therefore, cot/cot and cotl+can be discriminated by treatment of hyperthermia. Meanwhile, individuals with cot/+genotype showed cot phenotype at45℃, indicating that cot isn’t a completely dominant mutant. The cot mutant also showed the obvious cot phenotype in the neonatal larvae, larvae individuals (day3of fifth instar) pupae and moths when grown at high temperature. After treated with high temperature, individuals with cot/cot genotype almost lost the creep behavior, while Dazao (+/+) still has the ability of creeping. These results indicated that cot mutant was induced by high temperature. Therefore, cot is sensitive to high temperature and can provide good materials for studying the mechanisms of thermo-environment adaptability in insects.In addition, the two strains of cot/cot ok/ok and+/+ok/ok had heartbeats of56.15and54.05beats per minute (BPM) at room temperature, respectively. After treated at42℃for3min, BPM of cot/cot ok/ok and+/+ok/ok was increased to86.6and67.25, but the BPM of the cot/cot ok/ok individuals were much higher than that of the+/+ok/ok individuals. This showed that the heart rates of cot/cot ok/ok individuals are significantly increased.2. Positional cloning of cotIn order to study cot mutant at molecular level, the cot mutant gene by positional cloning was carried out. A female cot moth was mated with a male Dazao moth to obtain F1progeny (cot/cot♀×+/+♂).For positional analysis,1255individuals (418wild-type individuals and837cot mutants) were obtained from backcrossing generation using treatment of high temperature. By positional cloning, the cot locus was mapped within～268kb on the scaffold Bm_scaf3(chromosome15) between SNP19and SNP25by using1211BC1individuals. Ten genes were predicted to be present in this region, including BGIBMGA007797, BGIBMGA007730, BGIBMGA007796, BGIBMGA007731, BGIBMGA007795, BGIBMGA007732, BGIBMGA007733, BGIBMGA007734, BGIBMGA007794and BGIBMGA007735. Based on bioinformatics analysis, it was shown that BGIBMGA007794gene, a potassium voltage-gated channel gene, is able to mediate the voltage-dependent potassium ion permeability of excitable membranes. It possesses highly homologous with Drosophila seizure gene, which induced the similar phenotype with silkworm cot in Drosophila seits mutant. It was thus speculated that BGIBMGA007794was the mutant gene for cot mutant3. Identification of mutant site and functional verification of cot mutant geneTo evaluate whether BGIBMGA007794(termed Bmsei) is responsible for cot mutant, its expression in heads of cot mutant was detected by RT-PCR and the sequence was analyzed further. It showed that Bmsei has at least three alternative splicing isoforms in cot/cot and cot/+individuals. Compared to wild type, Isoform I completely lacked the fifth exon and caused a premature stop codon at733nucleotides, encoding an truncated protein lacking the fourth, fifth and sixth transmembrane domains, the P region and the cNBD domain; Isoform II had a36bp deletion in the3’-end of the fifth exon, resulting in a deletion of12amino acids in the fifth transmembrane domain; Isoform III contained the fifth intron and a premature stop codon at position of853nucleotides, without the P region, the sixth transmembrane domains and the cNBD domain. Genomic sequencing of Bmsei from the cot mutant showed a15bp deletion in the fifth intron and this deletion was unique to the cot mutant. This might result in alternative splicing of Bmsei to produce at least three abnormal transcripts. Tissue expression profile analysis showed that Bmsei was highly expressed in heads, anterior and middle silkgland and ganglia. And stage expression pattern analysis showed it was expressed from the late embryo to moth. For the verification the molecular function of Bmsei gene, the behavior of silkworm was observed by injecting potassium channel blockers. The result showed that wild type (Dazao) induced similar phenotype of cot exposed to high temperature when injected at various concentrations of potassium channel blockers tetraethylammonium chloride (TEA) and BaCl2, suggesting that the cot mutant gene is directly/indirectly linked to potassium channel protein. Bmsei expression was markedly decreased by RNAi through dsRNA injection during late embryo stage, leading to the cot phenotype of with shaking body, and vomiting gut juice, which is similar to the phenotype of cot treated with high temperature. This result indicated that Bmsei was responsible for cot mutant. Similarly, gentamicin at different concentrations was injected into cot mutant moths. For silkworm moths, the wings of cot mutants were nearly horizontal and resembled the WT with increased doses of gentamicin under high temperature. This result demonstrated that gentamicin could partly rescue the cot phenotype.4. Preliminary studies on molecular mechanism of cot mutantBy immunofluorescence staining, larval ganglia of+/+and cot/cot were isolated on the day of fifth instar. It showed that Bmsei was highly expressed in Dazao, and weakly expressed in cot mutants. To further determine the location of Bmsei, immunofluorescence of ganglia sections from day5of fifth instar of WT larvae showed the localization in the cell membrane and the cytoplasm. Immunoblotting showed that two bands of around100kD were detected in sample of wild-type (+/+), cot/+and cot/cot at room temperature and high temperature, and the expression of two bands was remarkably decreased in cot/+and cot/cot; the expression of two bands was decreased in all3strains treated at42℃, and was significantly decreased in cot/+and cot/cot compared to+/+, especially in cot/cot. These results showed that the synthesis of Bmesi was abnormal, which might cause seizure phenotype of cot mutant.5. Transcriptomic analysis of cot mutantTo investigate transcriptional changes related to the cot mutant when exposed to hypethermia, six DGE tag libraries were constructed, numbered as cot (cot/cot ok/ok)/25℃, cot (cot/cot ok/ok)/42℃, ok (+/+ok/ok)/25℃, ok(+/+ok/ok)/42℃, Dazao (+/++/+)/25℃and Dazao (+/++/+)/42℃. Compared to the untreated control group,147,221and24DEGs were detected in the cot (cot/cot ok/ok), ok (+/+ok/ok) and Dazao (+/++/+) strains after treated with high themperature, respectively. Among them,12common genes showed differential expression patterns in all of the three strains exposed to hyperthermia (42℃) for5min;30genes showed differential expression in both the cot (cot/cot ok/ok) and ok (+/+ok/ok) strains, and19genes showed differential expression in both the cot (cot/cot ok/ok) and Dazao (+/++/+) strains. Interestingly, in the12common genes,11up-regulated genes were annotated as HSPs or HSP related protein, and only one gene that encodes an annotated uncharacterized protein (LOC100216500) precursor was significantly down-regulated. Most HSPs were up-regulated in the3strains after treatment with high temperature, suggesting that they have important role in response to high temperature in silkworms. To identify DEGs related with hyperthermia in the cot mutant,110DEGs that were exclusively detected in cot were selected for subsequent analysis. Unique DEGs of interest were mainly involved in the ubiquitin system, nicotinic acetylcholine receptor genes, cardiac excitation-contraction coupling or Notch signaling pathway. These DEGs cause cot mutant to manifest the abnormal contraction of body segments and increase heartbeat under high-temperature regimes (>35℃). Insights into hyperthermia-induced alterations in gene expression and related pathways could provide hints for understanding the relationship between behaviors and environmental stimuli (hyperthermia) in insects.