Study On The Mechanism Of Naked Pupa

Naked pupa (Nd) of the silkworm Bombyx mori is originated from a natural mutation. The naked Nd gene and the fibroin heavy (H)-chain gene (Fib-H) had been found to be located at the 25th chromosome and closely linked. The early research results showed that the reason of naked pupa may be caused mainly at transcriptional level,but the molecular mechanism is still unclear. Mapping of Nd gene and studying on the occurring mechanism of Nd,not only good for the exploitation and utilization of silkworm resources and build a new type of silkworm bioreactor, but also can reveal the mechanism of silk protein expressed and secreted. These results can provide an important theoretical basis for silkworm silk too.Owing to a lack of crossing over in females, P50, a silkworm strain that spins normal cocoons, and Nd, a strain with naked pupae, were used as parents to obtain backcross populations P50×(P50×Nd) and (P50×Nd)×P50, designated as BC1M and BC1F respectively. Subsequently, mapping of Nd gene was conducted using the constructed simple sequence repeat (SSR) molecular marker linkage maps, and 7 SSR markers were found to be linked with Nd gene. By utilizing another BC1M population, we constructed a genetic linkage map for the Nd gene. The genetic distance of this map is of 96.8 cM with the Nd gene located at 60.8 cM. Meanwhile, 2 closely linked SSR markers, namely S2513 and S2511 which is 0.1 cM and 1.1 cM away from the Nd gene respectively were obtained. We analyzed the genome sequences from Kaikoblast database using the software. From the result we found that the Nd gene and fib-H gene are both in the Bm_scaf32,and the distance between Nd gene and the 3'of fib-H gene is about 159Kb; the distance between S2511 and S2513 is approximately 900Kb,but the distance is too long to clone Nd gene.Secondly, we analyzed the expression of three fibroin genes at the fifth day of fifth instar of 54A and Nd using quantitative real-time PCR method. From the results we found that the expression of fib-H in posterior silk gland of Nd silkworm at transcriptional level was greatly down regulated, it is about the 1/70 level of normal silkworm, while the expression of fib-L gene and P25 gene is 1/10 and 1/6 of that of normal silkworm. In order to verify the reason of it, we analyzed the expression of 3 silk gene controlling transcriptional factors using quantitative real-time PCR method. From the result we found that the expression level of POU-M1 in posterior silk gland of Nd silkworm is lower than in the normal silkworms. But the expression level of the other two factors SGF-1 and FMBP-1 is 1/2.48 and 1/8.44 of normal silkworms. Thus, the fibroin genes'abnormally expression is related to the deficient expression of POU-M1.