Mapping Analysis Of The New Mutant Purple Quail-like(q-l~p) In The Silkworm, Bombyx Mori

Epidermis is one of the most important tissues in silkworm, Bombyx mori, and it renovates after every time of molting during larval stages and is helpful for protecting the silkworm from being damaged. Marking is the most important character of epidermis and varies in shapes, colors, shades and so on. Therefore, researching on the marking has an important theory significance for pigment metabolism and marking formation mechanism.The purple quail-like( q-lp) silkworm is a new mutant that comes from the plain silkworm breed 932 VR. The q-lp mutant is extremely similar to quail mutant(q). The phenotype of the mutant q-lp is that the color of epidermis is light purple and there are markings on their epidermis while the wild-type has a normal skin color and no marking. Besides, the q-lp mutant takes little amount of mulberry leaves and shows weaker, develops slowly and unevenly and their larval body size and cocoons are smaller in comparison with the wild-type.Genetic analysis showed that the q-lp mutant is controlled by a recessive gene qlp. The preliminary studies showed that the mutant gene had located on the 8th chromosome by the test of morphological markers and SSR molecular markers. Based on these facts we had located the mutant gene using the skill of Map-Based Cloning. In order to identify the mutant gene, we had filtrated the genes at the target region by comparing their structures and expression profiles. These studies could lay a foundation for the further function study. The main research results were as follows:1. More SSR molecular markers were screened on the 8th chromosome to map the mutant gene q-lp and q-lp was located between two molecular markers nscaf2828-109 and nscaf2828-063. The genetic and physical distance between two molecular markers were 0.98 cM and 257 kb, respectively. There were 14 candidate genes in the area according to the silkworm genome database Silk DB.2. Epidermis of wild-type and mutant in 4th molting silkworms were used for semiquantitative analysis and 14 candidate genes had no difference in their expression level. Then newly-hatched silkworms were used for semi-quantitative analysis. The results showed that expression level of BGIBMGA005421-TA and BGIBMGA005422-TA in the mutant was lower than the wild-type. There were different annotations in two silkworm genome database Silk DB and KAIKObase. It had labeled two genes BGIBMGA005421-TA and BGIBMGA005422-TA in the database of Silk DB while there was only one gene whose function annotation was Orcokinin(abbreviate as Ork) labeled in the database of KAIKObase. The ORF of Ork gene included 5‘ UTR, the 1st and 2nd exons of BGIBMGA005421-TA as well as 3’ partial sequence of the 1st exon, the 2nd, 3rd and 4th exons of BGIBMGA005421-TA. The clone results demonstrated that the three genes BGIBMGA005421-TA, BGIBMGA005422-TA and Ork existed simultaneously.The ORF and genome clone of the three genes showed that there were no difference of BGIBMGA005422-TA between the wild-type and mutant and a single nucleotide deficiency in the 2nd exon of BGIBMGA005421-TA in the mutant compared with the wild type. The same nucleotide deficiency was also found in the mutant type of Ork gene3. Full length cDNA of BGIBMGA005421-TA gene in wild-type was 1099 bp including the 5‘ UTR of 67 bp, the ORF of 864 bp and the 3’ UTR of 168 bp as well as the Poly A tail of 28 bp. By BLAST in Silk DB we found that partial of 5’ UTR was separated by about 6 kb sequence upstream of the 1st exon. The core element of promoter TATA Box was found upon 20 to 30 bp of transcriptional start site. The BGIBMGA005421-TA gene expression profiles of various tissues by real-time quantitative PCR showed that it had the highest expression in fat body and also high in epidermis, midgut and weasand and other tissues all had low expression level. The expression profile of various development period showed that it was higher in glutonous stage than in other stages during larval stages and higher in 4th and 5th instars than in 1st, 2nd and 3rd instars. The expression level arrived to the highest peak in newly maturity stage.4. The deficiency of single nucleotide resulted in premature translation termination of BGIBMGA005421-TA and Ork, which could be the primary cause for the mutant. RNAi of BGIBMGA005421-TA gene in 2nd premolting silkworm of 932 VR showed that there were the black spot on the epidermis of the larva treated with dsRNA, which explained that BGIBMGA005421-TA gene closely related to the mutant phenotype of epidermis pigment. Ork, the myotropic peptide, was a kind of neuropeptide, one of whose functions was to promote the intestinal peristalsis. Mutation of Ork might slow down the intestinal peristalsis, which gave rise to obstacle of digestive ability. So it might be related to another character of q-lp mutant that took little amount of mulberry leaves, showed weak, developed slowly and unevenly and their larval bodies and cocoons were small.