| Due to the robustness of the male silkworm larvae, strong vitality of its pupae, high rate of cocoon layer they produced, long ,fine and evenly distributed silk fibers, the rate of silk produced by the male silkworm is about 20% more than that of the female; the grade of silk produced from silk fiber spinned by the male silkworm is raised to more than 2 grades than that of the silk fiber spun by mixed sexes, reaching to the level of 5A;what's more, the male silkworm produce quality cocoon and need 15% less of the amount of mulberry man the mixed sexes. For all the above-mentioned advantages in rearing of the male silkworm, one of the key measures in sericultural practice is the attempt to realize the monoculture of male silkworm, which will both raise the quality of me cocoons and reduce the cost for silk production. For these purposes, the selective breeding of male silkworm races has always been one of the focuses in sericulture of many sericologists home and abroad.Since the discovery of the high temperature lethality of the sex-linked chocolate (sch) during its embryonic stage and its sex-linked inheritance, new horizon has been open for the selective breeding of male silkworm races. Many researches have been done on the silkworm sch.However, up to the present the exact relation of the temperature sensitivity of sch to the sch gene itself on the molecular level is still uncertain. There were only sporadic studies on this and were based chiefly on RAPD techniques.Existing researches indicate: RAPD molecular marker as one of molecule means relied on PCR, can effectively and swift obtain genome information. So in genome research, we can use RAPD marker to investigate the purpose gene-sch gene through NIL in silkworm genome research.This research utilizes RAPD-PCR technology, and generously random amplify sch gene and its NIL, contrasting to find one steadily repeated sequence it linked with sch gene, i.e. OPD02. In order to obtain the information of this sequence further, cloning, sequencing and analyzing this gene fragment. The result showing:1.This section of fragment is a section of DNA sequence of coding SCP-x, named OPD02-759bp according to sequence result. Meanwhile, found it is part of the scp gene encoded SCP-x, i.e. the tail part of the gene coded the 3- ketoacyl-CoA thiolase.2. Designed the specific primers to amplify by PCR, sequencing and splicing, acquired genome DNA sequences coded 3- ketoacyl-CoA thiolase of the sch silkworm. The sequence ofcode district altogether is 1107bp, the proteins after translating are 369 a.a, and the molecular weight is 39.3kD.3. Analysized and compared die DNA and amino acid sequence of sch silkworm 3-ketoacyl-CoA thiolase. Result showed that there are 15 nucleoside acid differences with normal silkworm DNA sequence of 3- ketoacyl-CoA thiolase; mere are 5 amino acid differences after translated. Compared with normal silkworm and some other species to analysing, finding difference there is two amino acid mutation take place on the stabile structure district of 3- ketoacyl-CoA thiolase, and this mutation location is just the linked location of RAPD random primer, and discussed the perhaps function finally.4. Spliced Bmb007278 and Bmb021222 of the silkworm database, get the cDNA sequence coded SCP-x of silkworm, it is 1611bp altogether. The comparative result shows: Silkworm's SCP-x is 536 a.a, the molecular weight is 57.95 kDs, pI is 6.617, and che charge at PH7.0 is - 1.82. It is fewer 11 amino acid man the vertebrate (547a.a, 58 kD), fewer 8 amino acid man fruit fly (544 a.a). The result also shows that SCP-x N-terminus has the 3-ketoacyl-CoA thiolase activation, C-terminus has function structure domain of SCP-2.5. Utilized MegAlign of the software package DNAstar and aligned the SCP-x amino acid sequences of silkworm with those sequences, the phytogeny analysis are investigated and the phylogenetic trees are reconstructed based on the sequences, the result accords with the shap clusters. |
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