Fine Mapping Of 'Naked Pupa' Gene And Analysis Of Candidate Gene In Silkworm, Bombyx Mori

The'Naked pupa'(Nd) mutant of silkworm which was originated from natural mutation was initially found from strains of Europe by Nakano in 1951. The larva of Nd mutant can't spin a cocoon or spin very poor cocoon which only cotains sericin. The phenotype of Nd mutant can pupate normally and is hereditary. Four hereditary materials of Naked pupa have been found, which are Nd, Nd(2), Nd-s and Nd-s~D. The Nd gene and Nd(2) gene are linked with Fib-H gene which located on the 25th chromosome, however, the Nd-s gene and Nd-s~D gene are linked with Fib-L gene which located on the 14th chromosome.With the development of molecular biotechnolgy, molecular markers is widely applied in the silkworm. Molecular genetic mapping of silkworm lays a solid foundation for the positional cloning of genes. Xiaobo Yang constructed initial genetic linkage map of Nd gene using the constructed simple sequence repeat (SSR) molecular marker linkage maps, and 7 SSR markers were found to be linked with Nd gene. Meanwhile, 2 closely linked SSR markers, named S2513 and S2511 were found.To learn more about the function and mechanism of the Nd gene, we performed fine mapping of the Nd gene using SSR, STS and CAPS markers in the foundation of preliminary mapping, and 2 STS markers, 4 CAPS markers were found,named STS12,STS126,CAPS080,CAPS079,CAPS124 and CAPS029. By fine mapping, we succeeded in narrowing the candidate region to approximately 170 kb. No recombinant individuale had been detected by the marker CAPS29, which meant the Nd gene is located around CAPS29. The CDS and ESTs within the mapping region were blasted in SilkDB and KaikoBase. Mutation detection and Analysis of expression were accomplished by sequencing and semi-quantitative RT-PCR. Finally, a gene named G-1740 which was screened from candidate region was found to have alternative splicing between wild type and mutant. This gene may associate with mechanism of the Nd gene. Cloning of Nd gene and studying on the occurring mechanism of Nd would not only be good for researching of molecular mark assisted breeding, but also can build a new type of silkworm bioreactor and reveal the mechanism of silk protein expressed and secreted.