Semaphorin-1a-like gene plays an important role in the embryonic development of silkworm, Bombyx mori

Abstract

Fuyin-lethal red egg (Fuyin-lre) is a red egg mutant discovered from the germplasm resource Fuyin of Bombyx mori. The embryo of Fuyin-lre stops developing at the late stage of gastrulation due to chromosome structural variation. In this work, precise mutation sites at both ends of the mutated region were determined, and two inserted sequences with lengths of 1232 bp and 1845 bp were obtained at both ends of the mutation region. Interestingly, a bmmar1 transposon was detected in the inserted 1845 bp sequence. Bmmar1 possesses features of the Tcl/mariner superfamily of transposable elements (TEs), which belongs to class II TEs that use a DNA-mediated "cut and paste" mechanism to transpose. This finding suggests that Fuyin-lre mutation might be related to the "cut and paste" action of bmmar1. The mutation resulted in the deletion of 9 genes in the mutation region, of which the red egg gene re (BMSK0002766) did not affect embryonic development of B. mori, and the BMSK0002765 gene was unexpressed during the early stage of embryonic development. The RNA interference results of the remaining 7 genes suggest that the semaphorin-1a-like gene (BMSK0002764) had a major contribution to the embryonic lethality of Fuyin-lre.

Fig 1. Egg phenotype of Fuyin, Fuyin-lre, and Nistari at 72 h after egg laying.

(A) Wild-type silkworm variety Fuyin. The eggs are yellow when they are laid and then turn gray after 72 h due to diapause. (B) Fuyin-lre, red egg mutant of Fuyin. Some eggs turn gray and the other eggs turn red 72 h after egg laying. The gray eggs can hatch after incubation, but red eggs cannot hatch. (C) Diapause-free silkworm variety Nistari. The eggs are yellow during the whole egg period. The eggs laid by Fuyin are diapause eggs, and they need to be treated with hydrochloric acid to terminate the diapause. Silkworm eggs after injection are fragile, and hydrochloric acid treatment will cause the death of the eggs. The eggs laid by Nistari are diapause-free eggs and are widely used in microinjection experiments. Diapause-free eggs can develop normally without hydrochloric acid treatment. Therefore, the diapause-free eggs laid by Nistari were used in RNAi.

Fig 2. Structure diagram of the mutation region in Fuyin-lre.

In the Fuyin-lre mutant, the 281325 bp fragment starts from 11106950 bp and ends at 11388274 bp on chromosome 5 is replaced by some new DNA fragments. The 1232 bp and 1845 bp sequences were obtained at both ends of the mutation region by iPCR. 1–130 bp of the 1232 bp sequence and 1–186 bp of the 1845 bp sequence have multiple copies in the silkworm genome. 131–1232 bp of the 1232 bp sequence has only one single copy on the silkworm genome, which is derived from 11413374 bp to 11414475 bp of chromosome 5. A bmmar1 transposon was detected in 187–1845 bp of the 1845 bp sequence.

Fig 3. Identification of the insertion form of 131–1232 bp of the 1232 bp sequence.

(A) The position of primers used for PCR amplification; (B) PCR amplification results. PCR amplification could be performed when the Fuyin genome is used as a template but not when the Fuyin-lre genome is used. This finding indicates that 131–1232 bp of the 1232 bp sequence was inserted into the mutated region after cutting.

Fig 4. Relative expression levels of seven genes (BMSK0002759, KWMTBOMO02783, BMSK0002764, BMSK0002763, BMSK0002762, BMSK0002761, and BMSK0002760) in the eggs of Nistari at 24 and 48 h after RNAi.

The expression levels of BMSK0002759, KWMTBOMO02783, BMSK0002764, BMSK0002763, BMSK0002762, BMSK0002761, and BMSK0002760 were significantly decreased after RNAi. The BmActin3 was used as an internal control. Vertical bars represent the mean ± SE (n = 3).