| Our country owns the largest mutagenic resources of silkworm in the world. But for researching large-scale silkworm functional genome, it is far from enough, and majority of these mutagenic resources are the natural mutation. Thus, to provide abundant materials for functional research of gene, obtaining more mutagenic resources by artificial inducement mutation is one of the current important tasks. Previous research indicated that male is better than female during chemical mutagenesis induced effect in pupa and adult stage of silkworm. However, because of technological problems, it’s not success to finish induced mutation in larval stage so far. It’s essential to explore a method which could induce germ cells mutation of male silkworm in larval stage.As germ cells assume the special responsibility to ensure genetic stability of offspring, repair on gene mutation is more important in germ cells than in normal cells. And the formation of germ cells has meiosis which won’t appear in other somaic cells. Generally, it is believed that meiosis has effect on rejuvenation, which means there may be special and more efficient DNA repair mechanism, but there is no direct experimental evidence to prove it now.On this account, we investigated the effect of DES and NaNO2 on inducing mutant before and after meiosis of germ cells in male silkworm. On the other hand, utilizing in vivo and vitro experiments, adopted H2O2 to induce DNA damage of germ cells during different developmental stage, then detected repair ability and mechanism of male germ cells by single cell gel eletrophoresis and RT-qPCR before, during and after mitosis.1. Compared with control group, mutation group induced by 25-800μg/g DES had development delay inordinately, the dead rate increased, mating and laying rate after moths decreased, rate of dead eggs rised, mutation weren’t appeared in M1 generation, the abnormal larva and pupa were detected in M2 generation, but they cannot complete metamorphosis and death, so we didn’t obtain mutation progeny. Our results suggest that silkworm can be induced mutation and survival in larve stage after injected appropriate dose of DES.2. Silkworm can survival but exhibit obvious toxicity when 10-20 mg/mL NaNO2 was fed to silkworm in 3rd and 5th and dead when the the concentration is 40 mg/mL. Culturing and generating survival male silkworm, there is no mutation individual in the offspring. There remains deep research in the induced effect of NaNO2 on the silkworm.3. Injection of H2O2 to male silkworm in 3rd 36 h(most germ cells are at the stage of primary spermatocyte before meiosis)、4th 48 h(most germ cells are at the stage of meiosis)、5th 120 h(most germ cells have been completed meiosis to form spermatid and begin to deformation) and the sixth day of pupa(most germ cells have become sperm deformation) in order to DNA damage, the relative expression of APE in testis was measured. The result showed the influence of injection with H2O2 in different age was not an exact match. In addition to 5 ages, in control group injected with saline and low dose treatment group of other three ages, the expression of APE increased after 10-30 min of injecting, later decreased to the normal level. But in high dose group injection with H2O2 has little effect on APE. According to the results of this test, we speculate H2O2 through injection can’t access testis to effect on sperm cells and was broken down before this, thus it can’t cause the damage of DNA strand break of germ cells so as to trigger repair mechanism. However, the high dose H2O2 may affect the silkworm physiology seriously, even caused cell death such as severe injury so as to make DNA repair capacity decrease or lose.4. Germ cells from male silkworm testis of 3rd 36 h、4th 48 h and 5th 120 h are cultured in short-time in vitro and infected H2O2 with in order to induce DNA damage, then H2O2 was cleaned and further cultured, they are sampled in different time. We measured the situation of DNA damage and repair using SCGE. The results showed that the lowest of cell damage rate caused by H2O2 is sample from 4th 48 h, but three stages haven’t significant difference; but after cultivated 10 min in normal culture medium, the highest repair rate is germ cells of 4th 48 h, next for 3rd 36 h, damage rate of spermatid of 5th 120 h was higher than cells in 4 age; damage rate of germ cells in 3 age was significantly higher than those of other ages in 15 min and 20 min of repair; the vast majority of germ cells in different development time were all repired after 30 min of repair. These indicate for DNA damage caused by H2O2, repair capacity of most spermatocyte in the period of meiosis(4th 48 h) were strongest, next for after forming of sperm cells-5th 120 h, spermatogonium and primary spermatocyte of not yet meiosis(3rd 36 h) were the worst.5. The expression change of APE and DNA Lig after dealing with H2O2 were measured in vitro in 4th 48 h and 5th 120 h. The results showed that the expression change of APE and DNA Lig are basically the same in germ cells of 4th 48 h and 5th 120 h, their expression decreased within 30 or 60 min after contamination and rised after 60 or 90 min, but the expression of DNA Lig in 5th silkworm germ cells didn’t reach the level of no contamination. Compare with SCGE result, wo can find that the expression of APE and DNA Lig decreased instead of raising during repair of germ cells, and the increasing time obviously lag, and increasing range is small. Thus we assume DNA damage may exist other repair mechanism in the process of germ cells from spermatocyte to spermatogenesis. |
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