| It is discovered that the silkworm embryo of sex-linked chocolate (sch) possesses temperature sensitivity in its later development stage. Sex-linked chocolate (sch) hatched scarely when incubated under high temperature and dry condition, whereas the embryo is still alive. The stage which is most sensitive to high temperature is trachea colouring embryo(ji-3), while the stage sensitive to dry condition is body pigmentation embryo(ji-5), so these two development stages have been called high temperature sensitivity stage and dry condition sensitivity stage respectively. The gene of high temperature and dry condition sensitivity is defined as sex-linked temperature sensitivity gene(t), which exists in 21.5 location of Z chromosome linking closely to sch gene, and presents concealed heredity. This phenomenon exists in other races, but the molecular mechanism of temperature sensitivity is unknown. This research was carried out to explore the molecular mechanism of not-hatching embryo by analyzing the protein of embryo with temperature sensitivity under different incubating condition with proteome technology.By comparing two protein extraction buffers and six protein solubilization buffers consisting of different chaotrope, sulfobetaine and reducing agents, it is apparent that the best method to extract embryo protein is by using phosphate buffer and the solubilization buffer containing 8M urea,2M triourea, 4%W/V CHAPS, 20mM Trisbase, 30mM DTE, 2% carrier amphotytes (pH 3.5-10) to solve the protein of embryo.By MALDI-TOF-MS analysis and homology searching, it was found that the main specific protein in positive and negative crossing generation when crossing the race with sex-linked temperature sensitive gene and normal gene races as parents under high temperature and dry condition are homologous to Bombyx mori 6G1 gene for 3OK major plasma protein. In the positive generation, protein spot 6 and 8 are homologous to the 6G1 gene, while in the negative crossing generation, protein spot 6' is homologous to the protein sequence which is conferred by the Bombyx mori cDNA clone wdv30370 T3 " $ " named Bombyx. mori wing disk C108 5th-instar day-3 larvae., and protein spot 8' is also homologous to the 6G1 gene. Synthesizing the variety of protein spot 6 and 8, thus it is considered that embryo need to consume the major protein to synthesize the functional protein ahead and quickly. But at the same time ,the synthesizing activity of enzyme which is used to consume the major plasma protein normally is effected by the incubatingenvironment of high temperature and dry condition. Thus, the absorbing route of the major plasma protein is changed or cut, and the regular route of the amino acid application is also changed. It can not satisfy the need to synthesize the functional protein under high temperature and dry condition. Even if the major plasma protein is mostly consumed finally, but the embryo still can not resist the unfourable environment because of the lag time of the functional protein synthesized. The accumulation of major plasma protein in the embryo results in insufficient amino acid to synthesize the functional protein, and makes the mechanism of embryo development incomplete. It is also considered that the accumulation of protein 8 at last exists in the female pasent of the crossing generation with the temperature sensitivity gene race as male parent, which is the reason of not hatching..For further analyzing the protein variety and to understand the mechanism of high temperature and dry condition, the embryo is separated from yolk, egg shell and serosa to extract protein. Also 2D-PAGE patterns of embryo protein were achieved. There are 30 specific proteins between three different condition: normal temperature and humidity, high temperature and dry condition, high temperature and wet condition from the shortening stage to body pigmentation stage. It can be considered that embryo come into being cooperation mechanism accordingly for resisting the bad incubating condition under high temperature and dry... |
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