Structure And Expression Pattern Analysis Of CBP Gene With Carotenoids Difference In Natural Colored-cocoon Strains Of Bombyx Mori

The domesticated silkworm, Bombyx mori serves as a model for Lepidoptera. The processes of pigment absorption, modification and transportation are under control of complex gene regulations. Mechanisms surrounding cocoon coloration regulation in Bombyx mori have become a main focus in animal studies for regulation of pigment absorption. Bombyx mori is an insect of economic importance in industry scale for silk production where white cocoons are more commonly used. Colorful silk-related clothes come out of chemical painting procedures, resulting in large quantity of polluted water. Development and usage of strains with natural colored-cocoons can give vigor to silk industry where production process is friendly to environment. Understanding the molecular mechanisms about pigment absorption and transportation processes will give us a closer view of the pigmentation and regulation theories in animals including us human. It is also positive as to benefit breeding of natural colored-cocoon strains.Carotenoid binding protein gene (CBP) has been found to be a key gene involved in yellow cocoon coloration where the mRNA expression difference destined by distinctive gene structures contributed an important factor to different cocoon colors. To date, there are few reports concerned about CBP gene structure and expression comparison among strains with different cocoon colors. The developmental pattern of CBP gene transcription also has to be elucidated. Together with specific investigation of carotenoids distribution in strains of different cocoon colors these studies can provide base for the construction of descriptive molecular data to each strain.Therefore this study was carried out to investigate and analyze in detail CBP gene structure and development transcription pattern among strains of different cocoon colors and their relationship to carotenoids distribution, using methods including bioinformatics, PCR, RT-PCR, Southern blotting, UV-Vis scan and RACE technology, etc. The results showed that:1. There are at least four different CBP gene types in Bombyx mori, among which two to three types can be found in yellow cocoon strains and only one type with sequence difference can be found in white or green cocoon strains, leading to expression patterns in yellow cocoon strains distinct from those in white or green cocoon strains. CBP gene transcription exists in all the developmental stages under investigation including larvae and pupae period of Bombyx mori and the transcription pattern was different respecting to cocoon colors, developmental stages and tissues. CBP mRNA expression pattern in strains with yellow cocoon was different mainly in transcript types and sequence from those in white or green colored-cocoon strains. CBP mRNA expression in the silk glands was repressed in the fourth stadium of larvae in contrast to other tissues like in the gonad where normal CBP mRNA expression was observed.2. Different from published literatures, CBP gene in strains with yellow colored-cocoons of Bombyx mori like N4 was transcribed simultaneously in an array of products with highly conserved sequence. This transcription pattern may not be limited by development stages and the exact transcripts could differ in sequence among strains.5' UTR was found to be the main region that distinguished these transcripts. Missing of large areas was also observed including in the coding sequence. Similar transcription pattern was not found either in white colored-cocoon strains like C108 or in seven other strains with green cocoon where only one transcript was observed in all development stages under investigation. The difference in CBP mRNA expression pattern could come from distinctive CBP gene structures among strains with different cocoon colors of Bombyx mori.3. Other alternative transcription products were also identified from CBP gene in strains like N4 with yellow colored-cocoons of Bombyx mori. These transcripts were distinguished mainly in the 5'UTR and the ORF of these transcripts showed high conservation with CBP only the sequence length was longer in the latter one. The same regulation mechanism may pose on expression of all the transcripts from CBP gene. Two of the thirteen alternative transcripts identified in this paper were truly expressed in all strains under investigation.4. Absorption difference of carotenoids was observed among investigated strains of Bombyx mori according to their cocoon colors. UV-Vis spectrum analysis showed that seven strains with green cocoon and two strains with white cocoon could not absorb carotenoids, suggesting a possible relation to truncated CBP mRNA expression in the main carotenoid absorption tissues in these strains. Carotenoids absorption was developmentally regulated with tissue distribution difference in twelve strains with yellow colored-cocoon of Bombyx mori. Similar carotenoids distribution pattern was found in the fourth stadium of larvae in strains with yellow colored-cocoon of Bombyx mori. Specially, carotenoids content in the silk glands was negligible in the fourth stadium which may come from repressed CBP mRNA expression during this development period. Carotenoids content in the fifth stadium of larvae was significantly higher than those in other development stages. Distribution pattern difference was also observed among strains in the fifth stadium of larvae reflecting distinguished developmental characters and cocoon color diversification but not carotenoids absorption ability.Combining all these data a conclusion was made that CBP mRNA expression pattern difference among colored-cocoon strains of Bombyx mori may come from distinctive gene structures in yellow cocoon strains from those in strains with other colored-cocoons which may influence carotenoids distribution pattern and further cocoon colors. Structural features of CBP gene could contribute largely to this complex transcription pattern in CBP gene site, suggesting Bombyx mori could serve as a good animal model in complicated gene expression and regulation investigations and CBP gene may show effective in pigment absorption and gene expression studies.