| Biological body coloration is a very common phenomenon, and each individual has its own unique pigmentation pattern different from the same species and other species. Coloring has many biological functions, such as adaptation to the living environment, avoiding predators prey, participating in its own development and ultraviolet protection. Insects (larvae and adults) have a rich body color and pigmentation pattern, and the study of molecular mechanisms of insect coloration found that, the mutation of pigment biosynthesis related genes and upstream regulator gene will produce a new color pattern and increase coloring diversity. However, in people, mutation of the pigment synthesis pathway genes can also cause human color changes in hair, eye color, and skin, often accompanying with the related disease.Insects are the most prosperous organisms in biosphere, and they own rich diversity on body color. So far, the understanding of the molecular mechanism of insect body color is still very limited. The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm(Bombyx mandarina) about 5,000 years ago. As an important model insect, silkworms are rich in color diversity the same as butterflies, fruit flies and other insects. The completion of silkworm genome framework map and a series of major research project provide abundant data for studying the molecular mechanism of silkworm body coloration. Moreover, there are about 1000 mutants maintained in silkworm gene resource bank at Southwest University (Chongqing, China), many of which are about body color. Therefore, silkworm can be used as a good model for the evolution of biological coloration and human disease.In this study, we used the two silkworm strains 961B and 961C as research material.961B and 961C are silkworm strains originated from hybridization of domesticated silkworm (excitation China) and the wild silkworm in the spring of 1996, and the body color of 961B silkworm larvae is white, whereas the pigmentation pattern of 961C silkworm larvae has significant changes from first instar to fifth instar. More definitely, the body color of 961C is mainly black before the 4th molt, with white stripes in the two sides of the eye spot and the tail. From the 4th molt period to the start of 5 instar, the body color changed significantly, showing a brown body, with a light red skin close to the head and other position. However, there are no white coloration in the fifth instar. With the growth of silkworm, the body color is lighter, and more close to, even the same as the color of the bark of mulberry trees. We used transmission electron microscopy, high performance liquid chromatography and molecular biology techniques, in cell, physiological, biochemical and molecular level up to compare two different silkworm strains of skin color, to study the molecular mechanism of body color of silkworm. Main contents and results of experimental study are as follows:1. Comparison of epidermal ultrastructureThe epidermis of 961B and 961C at day 1 of the 5th instar were fixed, stained, sliced and observed by transmission electron microscopy. The result showed that epidermal cells of 961B contained large quantities of uric acid salt particles, and the outermost layer of the cuticle had no black material. In 961C, the epidermal cells contained less uric acid salt particles than the 961B, and there were many ommochrome granules with uniform size and different color; moreover, melanin particles existed in the cuticle.2. Analysis of melanin precursor contentWe extracted epidermal melanin precursor mixture from the integument of the 961B and 961C at the three main periods (fourth instar three days, fourth moltting and fifth instar one day), respectively. Three precursor substances (dopa, dopamine and N-P alanyl dopamine) were analyzed by high performance liquid chromatography (HPLC). The results showed that:(1) as the melanin synthesis pathway upstream material, dopa contents of the epidermis are not different between the two silkworm strains, but the content of dopa in the last period is twofold more than that in the first two periods. (2) dopamine contents between the two silkworm strains showed significant differences, dopamine in 961C was higher than that in 961B at the three periods t, and the content in the 4th molt was higher than that in the other two periods. (3) N-βalanyl dopamine contents of the epidermis in the two silkworm strains were increasing at the three periods, and the NBAD content of 961C is higher than that in 961B.3. The expression profiles of pigment synthesis pathway genesThe expression profiles of pigment synthesis pathway genes were analyzed in 9 tissues and 10 periods of 961B and 961C By RT-PCR and qPCR. The results showed that the two strains had similar tissue expression profiles for pigment synthesis pathway genes except for BmYellow-f, BmKynu and BmGTPCHI.961B and 961C also had the similar developmental expression patterns, but the amount of expression of 961C was much higher than that in 961B,961C after 4th molt. The expression of BmTH, BmDDC, BmDat expression increased significantly after 4th molt.The experimental studies show that biological skin coloring is jointly determined by multiple genes. Through the cell, physiological, biochemical and molecular level studies, we found that uric acid particles content in the epidermis of silkworm was different; BmYellow-f, BmKynu and BmGTPCHI had differences in tissue expression, and the expression of BmTH, BmDDC and BmDat increased significantly, BmTH may be regulated by cis-regulatory elements, which makes the significant difference on the body coloration. This study establishes foundation for further study of molecular mechanism of silkworm coloration. |
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