温度对家蚕缩蚕突变表型产生的影响及cot基因的初步定位

The Impact of Temperature on Producing Contractile Phenotype and Primary Mapping of cot Gene in Silkworm,Bombyx mori

缩蚕(cot)是家蚕的行为突变体之一,其表型由单基因控制,呈隐性遗传。在室温下对缩蚕突变体幼虫的体表反复摩擦,蚕体出现微弱的收缩,但摩擦停止即恢复正常;将缩蚕突变体幼虫置于温度>30℃的环境5 min以上,可呈现明显的缩蚕表型性状,表明缩蚕突变体对温度极为敏感。进一步分析基因型为cot/cot、cot/+的家蚕5龄幼虫对温度的敏感程度:cot/cot基因型家蚕经30℃以上高温刺激5 min即产生明显的缩蚕表型性状;cot/+基因型家蚕经45℃高温刺激10 min可出现微弱的缩蚕表型性状;正常型(+/+)品种大造即便经48℃高温刺激10 min也未见异常表型。因此,通过温度处理可以鉴别cot/cot和cot/+的基因型个体。以cot/cot(♀)×+/+(♂)获得F1,cot/cot(♀)×F1(♂)得到BC1M群体,分别以通过高温处理判别的正常型和突变型个体作为定位群体。首先利用8个SNP标记和96个BC1M代个体构建低密度连锁图谱,cot突变基因位于15号染色体标记15034_16.4~15100_27.2区域内,遗传距离为10.8 cM。再利用17个SNP标记和1 115个BC1M代个体构建高密度连锁图谱,初步将cot突变基因锁定在nscaf2888_1268334和nscaf2888_1000396之间268 kb的区域内,其间有10个候选基因,功能注释其中有5个基因都与神经信号的传递有关。

The contraction mutant （cot） is one of the behavioral mutants in the silkworm, Bombyx mori. Its phenotype is controlled by a single gene and inherited in the recessive pattern. At room temperature, larvae of the cot mutant showed slight contraction when their bodies were repeatedly rubbed, but became normal immediately after the rubbing ended. After being placed under over 30 ℃ for over 5 min, larvae of the cot mutant displayed obvious cot phenotype, demon- strating that cot mutant is especially sensitive to temperature. Further analysis on the sensitivity of cot/cot and cot/ ＋larvaeof the 5th instar to temperature revealed that, the cot/cot larvae displayed obvious contraction when treated under over 30 ℃ for 5 min, the coW＋larvae presented slight contraction when treated under 45 ℃ for 10 min, and the normal silkworm variety Dazao （＋/＋） did not show any contraction even under 48 ℃ for 10 min. Therefore, cot/ cot and cot/＋ genotypes can be discriminated by treat- ment of high temperature. Consequently, cot/cot （ ♀ ） was crossed with＋/＋（ ♂ ） to obtain F1 progeny, cot/cot（ ♀ ） was crossed with FI（ ♂ ） to obtain BC^M population, and the normal silkworm variety and the cotmutant were used as mapping populations respectively. Firstly, a low-density linkage map was constructed with 8 SNP markers among 96 individuals of the BClM generation, which showed that the cot mutant gene was confined to a genetic distance range of 10.8 cM on chromosome 15 between two SNP markers, 15034_16.4 and 15100_27.2. Next, a high-density linkage map was constructed using 17 SNP markers and 1 115 individuals of the BC_ M generation, which narrowed the region harboring cot to -268 kb between nacaf2888_1268334 and nacaf2888_1000396, which contains 10 predicted genes. An examination to functional annotation revealed that 5 of the 10 genes are related to transduction of neural signal.