Proteomic Analysis Of Silkworm Testis And Male Accessory Glands And BmsHSP20.2 Research

The silkworm, Bombyx mori, is an important economic insect and also a lepidopteran model system. It can be used for the study of organ differentiation, growth development and regulation, genetic variation of organisms. Silkworm testis is a physiology organ to produce sperm, in which the formation of sperm cell experience 4 periods, including proliferation, growth, maturity, and deformation. The maturated sperm will be mixed with accessory gland secretions to form semen. The accessory gland secretions could activate sperm and provide energy to sperm.During sperm maturation process, it was affected by many external factors, such as temperature, pH, hunger, osmotic pressurefor; In addition, different genetic strains of their reproductive condition is different, but the mechanism is not clear. In view of this, the experiment mainly through proteomics method analysis on fertilization rate of different silkworm strains and two conditions treat of Dazao, found out the differential proteins, then carried on the qualitative analysis; and the proteomics of testis and male accessory gland were also analyzed using proteomic approach to high temperature and starvation conditions treated silkworm of the final analysis, screening to follow-up studies of protein, and carries on the thorough research, the main research results are as follows:1. Proteomic analysis of silkworm testis with different conditions treatBy proteomic analysis found that three protein spots more obvious changes in different strains of 16-500 (fertilization) strains and two conditions treat. The three proteins were glutathione S transferase (BGIBMGA009107), small heat shock protein 19.8 (Choristoneura fumiferana BGIBMGA005784), small heat shock protein 19.9 (BGIBMGA004540)2. Proteomic analysis of silkworm moth male accessory gland in 1 daysUsing the same method, we analysis male accessory gland and high temperature treatment of male accessory glands in silkworm.The first, proteomics analysis of male accessory glands in silkworm, as male accessory gland tissue, which adhesion amount of body fat, so we used fat body as a control, eventually found have four protein spots is high quantity expression in male accessory gland tissue, and by MALDI-TOF-MS identified. The four protein spots gene numbered are:BGIBMGA009423, BGIBMGA000168, BGIBMGA001815, BGIBMGA014604. Through information analysis and found that the four protein domains are unknow, and no related to functional studies. We further analysis and found that the four genes are specific expression in different organizations and period by microarray data and RT-PCR. Other, we found it have changes in the protein level with high temperature stimuli, but unidentified succeed.3. BmsHSP20.2 gene cloning and prokaryotic expressionWe filter during in differentially expressed proteins, and finalized BGIBMGA005784 (BmsHSP20.2) gene as a follow-up study of gene. BmsHSP20.2 is a small heat shock protein, there is no function has been reported.The gene is specific expression in testis tissue, and high levels of expression in the pupal stage by microarray data and RT-PCR expression analysis in silkworm, To further study the function, we successful cloning and prokaryotic expression.4. Tissue localization of BmsHSP20.2The result shows that the gene in testis of silkworm specifically expressed by microarray data and RT-PCR express spectrum analysis, in order to further test the result, we used western-blot experiment, found that the result is same with chip data and RT-PCR express spectrum analysis in testis specifically expressed, and is gradually reduce in day-5 of fifth instar to moth period, this period of sperm into the deformation gradually mature sperm. Also, by immunofluorescence positioning experiments, will BmsHSP20.2 positioning in sperm in the beam that is in BmsHSP20.2 may expression in sperm cells. The results show that the formation of the BmsHSP20.2 may and sperm inevitable connection.5. BmsHSP20.2 stress and chaperone function verificationThe results show of Western-blot that the BmsHSP20.2 expression was increased by high temperature and starvation treatment, the description BmsHSP20.2 have stress.Other, Lysozyme induced by DTT was further validate the BmsHSP20.2 have molecular chaperone function.