| The fluorescence color of silkworm cocoon is related to the sex, we have succeeded in breeding the sex-linked cocoon fluorescence color varieties, including Chinese strain"Yingguang","Yingxian 8417"and Japanese strain"Chunyu", by the fluorescence sorting of cocoon and directive breeding etc methods. F1 cross combination "Yingguang×Chunyu" has passed varieties examination and been approved by the national special silkworm, this is the first time both at home and abroad. However, the genetic mechanism of the sex-linked and fluorescent pigment metabolism differences between male and female and so on are not clear yet. Solving these questions will have important academic and practical value both in richening silkworm fluorescent pigment metabolism genetics theory and guiding the breeding practice of the sex-linked cocoon fluorescence color variety and so on.We studied the cocoon fluorescence color characteristics, and focused on the sources of fluorescent pigment in the sex-linked cocoon fluorescence color varieties\male and female differences in metabolism\genetic mechanism and molecular markers etc, the main results were as follows.1. The cocoon fluorescence color of varieties keeping in our institute could be divided into the "yellowish white cocoon fluorescence color", " bluish purple cocoon fluorescence color" and " mixed cocoon fluorescence color " three types; for the same silkworm variety, the cocoon fluorescence color of male tended to yellowish white and female tended to bluish purple clearly; silkworms of the same variety cocooning under the environment of high temperature and low humidity, the cocoons with yellowish white fluorescence color were more, and under high temperature and humidity environment, the cocoons with bluish purple were more; the water solubility of sericin of cocoon with yellowish white fluorescent color was faster than the bluish purple; the sex-linked cocoon fluorescence color variety could be bred by hybridizing female with blue fluorescence color and male with yellow, and breeding for years.2. The sex-linked cocoon fluorescence color variety was reared by the artificial diets, which were prepared by mulberry leaf powder and mulberry leaf powder extracted with 70% ethanol respectively, from the newly molted silkworm or the third day of the fifth instar. The results showed: Feeding by the artificial diet with untreated mulberry leaf powder, the female and male cocoon could be distinguished under ultraviolet light; however both the fluorescence cocoon color of female and male were even purple when feeding by the artificial diet with mulberry leaf powder extracted with 70% ethanol, and it was unable to distinguish the female and male through cocoon fluorescence color. In one hand this showed that the basis material of fluorescent pigment resulting in the cocoon fluorescence color difference between male and female stemmed from the alcohol solubility material in mulberry leaf; on the other hand the difference between male and female cocoon fluorescence color might be appeared after the third day of the fifth instar. At the same time, it proved that such differences were determined by yellow pigment, so we decided to study it as the main fluorescent pigment.3. Both the results of the paper chromatography and HPLC analysis showed that the fluorescence pigment types had no significant difference between male and female. It was the different value, especially the yellow fluorescence color, causing the difference of female and male, the difference was in quantity not in quality; results of mutual transplanting the female and male silk gland confirmed that the fluorescence pigments of middle silk gland came from the blood directly, the middle silk gland had no selective absorption and permeability to the fluorescent pigment.4. In addition to the fluorescent pigment originated from middle silk gland, there was a kind of purple fluorescent pigment with the Rf value of 0.77 in the cocoon, both of the above didn't test from blood and middle silk gland. It showed that there was new change of fluorescent material in cocooning process, this might be one of the main reasons of the silkworm cocoon fluorescence color was easy to be influenced by the mounting conditions.5. The yellow fluorescent pigment which played decisive role in distinguishing the female and male by cocoon fluorescence color would be appeared after the fourth day of fifth instar. As follows: after the fourth day of fifth instar, the fluorescence colors of female and male in blood, middle silk gland and cocoon were purple and yellow respectively with increasing days. On the contrary, yellow fluorescence color in the female midgut deepened continuously and it was bright yellow from the fifth day of fifth instar to mature silkworm, however the main part of male midgut was brown during the entire fifth instar. It showed that the yellow fluorescence color of male in midgut could be formed, penetrated and released into blood successfully. However in female the synthetic had no problem, it couldn't be penetrated and released into the blood smoothly, accumulated in midgut instead. It was the main reasons for the difference between male and female cocoon fluorescence color that yellow fluorescent pigment could not be penetrated through the female midgut smoothly leading to the reduce of the total amount of yellow fluorescent material in the female blood clearly. We made a reasonable explanation on the theory to the cocoon fluorescence color sex-linked phenomena of purple in female and yellow in male and to another phenomena of the male tending to yellowish white and female tending to bluish purple in the same variety.6. This type of fluorescent pigment had good solubility in the solvent of moderate concentration of alcohol, but poor in the high concentration. The results of HPLC analysis were: using methanol as the mobile phase, it could be eluded down early, and the absorption peaks appeared earlier. The polarity of these components was high and hydrophilic. We knew the molecular weight of the pigment was 453 by the separation, purification and mass spectrometry analysis of fluorescent yellow pigment, and it might contain flavonoids pigment with a five-carbon sugar.7. Study on hybrid genetic indicated that the yellow fluorescent pigment, which played decisive role in distinguishing the female and male, were quantitative genetic, and controlled by multiple genes, the genes which controlled this character were mainly on regular chromosome; The reason that caused different cocoon fluorescence color between male and female of fluorescence sex-linked varieties was that the yellow fluorescent pigment in male silkworm cocoon was more than female significantly. This difference was that this kind of pigment in the female could not penetrate through the midgut effectively, compared to the male, but accumulated in the midgut. There were two possible reasons, the first might lack of small transporters, and the second might combine with a substance, so could not penetrate through the midgut. Because that male silkworm did not have this problem, so both reasons were related to the W chromosome which determined the female, it was one kind of gene on W chromosome brought the result. Therefore, although differences showed in the male, but the basic reason was on the female, so the genetic of the sex-linked cocoon fluorescence color should belong to the genetic type of sex-limited.8. With the large number of molecular markers, the results showed that molecular marker ISSR-11 was linked to the fluorescent sex-linked and amplified bands in the molecular weight of 1541 bp sites. "Yingguang", "Chunyu," the two silkworm varieties with the sex-linked cocoon fluorescence color had amplified a specific band in the male silkworm, but females in the same site with the specificity of the very shallow. Previous studies had proved, the main reason of causing differences between female and male of the sex-linked cocoon fluorescence color varieties was the different of yellow pigment quantity in female and male (more in male, less in female). ISSR-11 primers amplified bands of the male and female differences could analysis the characteristics of fluorescence cocoon color sex-linked reasonably. It could use ISSR marker for the difference between male and female of the sex-linked cocoon fluorescence color silkworm varieties. |
