Study Of Different Expression Genes In The Yellow Cocoon Near-isogenic Lines Of The Silkworm, Bombyx Mori

In the silkworm, Bombyx mori, the pigments in yellow cocoons are derived from carotenoids which are absorbed from mulberry leaves and transferred to the silk gland via the hemolymph. They are controlled by three genes, yellow blood (Y), yellow inhibitor (I), and out-layer yellow cocoon (C), which are involved in carotenoid uptake and transport. The Y gene controls the uptake of carotenoids from the midgut lument into the midgut epithelium, while the C gene controls the transfer of carotenoids from the hemolymph to the silk gland, the I gene down-regulates the yellow colring of the midgut epithelium into the hemolymph.Silkworm near-isogenic line (NIL) of yellow cocoon (C) gene were constructed using silkworm yellow cocoon strain KY and white cocoon strain C108. The NIL of yellow cocoon were bred through backcrossing successively using KY as donor parent and C108 as recurrent parent. The method of morphologic comparability and the technology of SSR marker were introduced into the polymorphism analysis and the near-isogenic level evaluation on the NIL. The results indicated that the yellow cocoon NIL can be propitious to be used in the next study of the C gene, the high quality NIL can be easily developed and the near-isogenic level can be objectively evaluated by the combining utilization of classical methods and modern molecular marker technologies.Individuals with yellow and yellowish middle silk glands were selected from yellow hemolypmh individuals among the yellow cocoon NIL population of the 11th backcross generation and were used to construct the subtracted cDNA library using PCR-SelectTM cDNA Subtraction Kit (Clontech). Plasmids were extracted from 104 positive clones randomly selected from the library and then sequenced and assembled using CAP3 software. As a result, 16 deduced genes (contigs), 25 ungenes and 5 unknown genes were obtained. BLASTx searches and homology analysis with exising expressed sequenced tags (ESTs) revealed that these genes were involved in many biological processes such as energy metabolism, gene transcription, signal transduction, substance transportation, cell growth and division, protein synthesis or degradation, nucleic acid and protein processing, and organismal defense etc. Real-time quantitative PCR analysis to the relative expression level of differentially expressed genes which selected from SSH cDNA library in different developmental stages of the NIL's 5th instar individuals with yellow and yellowish hemolymph individuals respectively revealed that the expressed level of these differentially expressed genes may have a close relationship between these genes and the yellow cocoon phenotype in silkworm. Our results provide useful fundamental information for further studies on molecular mechanism of color formation in silkworm cocoons.In order to acquire basic information of related proteins which function synergistically with silkworm yellow cocoon gene, we also employed two-dimensional polyacrylamide gel electrophoresis and ImageMaster 2-D platinum approach to separate and analyze middle silk gland proteins of the 4 day 5th instar silkworm larvae with yellow and yellowish middle silk gland of yellow cocoon NIL. Distinct protein spots on silver-stained gel were mainly distributed,and the matrix-assisted laser desorption ionization and time of flight mass spectrometry (MALDI-TOF-MS) was applied for identifying major protein spots on synthesis. 6 spots were identified to involve in many biological processes including energy metabolism, transmembrane transport and immue-associated protein etc. These proteins are probably related to the formation of silkworm yellow cocoon character.