Construction Of The Molecular Linkage Map And Isolation,Identification And Particular Expression Of Small Heat-shock Genes In Silkworm,Bombyx Mori.

Silkworm(Bombyx mon.) is an important economic insect and an ideal model organism for the biology study. The morphologic genetics of silkworm has been studied for more than 80 years, and over 300 genes for morphological traits have been localized on the traditional genetic linkage map. On the development of molecular biology, a lot of progress has been made on the molecular genetics of silkworm in many countries such as Japan, America and China. Those studies related to gene cloning, gene location and molecular breeding are sure to become the major research field for silkworm genetics. Construction of the molecular linkage map may greatly promote the molecular biology research in silkworm. Furthermore, it is also an important bridge between basic and applied research in silkworm. It can carry out the analysis and location of QTLs for quality or resistance to diseases. It may make an efficient breeding possible through the selection of molecular markers, and it may also carry out the research of cloning genes and introduction of foreign genes. The following are the major content of the paper: On the basis of the establishment of the method of screening RAPD markers, the first molecular linkage map of silkworm in our country was constructed; FDD(Fluorescent differential display) was used to study the gene expression of embryonic lethality of ki and normal egg during the early embryonic stage at mRNA level and to separate the target gene and clone it. Two small heat shock genes, Bmhsp20.8A and Bmhsp20.8B, were cloned in the FDD analysis of the embryonic lethality of ki. And the expression of sHSPs genes was studied in the silkworm, and their functions were analyzed. Simultaneously systematic evolution of shsps in the silkworm and other species were analyzed, the results showed that the directly homologous evolution of shsps accounted for the major, but the simultaneously homologous existed too. I.Screening for the molecular markers Random amplified polymorphic DNA, which was known as RAPD, was used to screen for the DNA markers of silkworm. Using the RAPD, a marker could be amplified by a single primer (normally 10 bases) at low annealing temperature when there is a sequence on opposite strand no more than 3000 base pairs in an inverted orientation. The RAPD method allows a rapid identification of DNA markers and provides an efficient assay for polymorphism. A large amount of RAPD markers were obtained using the method to screen the silkworm genome. 1.1 Reaction conditions of RAPD are different in different species. The optimized RAPD reaction conditions suitable for silkworm were established for silkworm genoniic DNA in the study. 1.2 With 477 RAPD primers of which each contain 60%?0%GC, the RAPD markers in C108,Dazao and F1 of Dazao x C108 were screened. Among the 410 of 477 R.APD primers we identified 5155 amplified DNA fragments. The average number of amplified DNA fragments was 12.6, varying from 1 band to 23 bands. Among them 1496 amplified DNA bands (29.0% of the total amplified fragments) showed polymorphism with a mean number fragments of 3.6 per primer. Among the 477 primers 67 of them produced no bands with the percentage of 14.1, 11 primers which showed no polymorphism with the percentage of 2.3, and the rest 83.6% could produce bands showing polymorphism. 1.3 The genetic pattern of RAPD serves as the basis for localizing genes and constructing the molecular linkage map. The study shows that RAPD markers have the pattern of dominance and recess...