| Macrobrachium nipponensis is an important freshwater species with high economic profits in China, which is also called freshwater shrimp. Gynogenesis is an important genetic technique which plays a role in establishing genetic identity genealogy and researching the sex determination mechanism. Gynogenesis is also a significant breeding technique, especially used in controlling sex. Gynogenesis is a developmental pattern, and in this pattern, sperms can enter into eggs while chromosome of sperms will disappear soon, and the development of embryos is totallygenetically inherited from the females.So far, researches have focused on gynogenesis of fishes, while there is almost no research on crustacean. As crustacean has unique broody features and sperms are coated inside spermatophore, there is no breakthrough of gynogenesis in crustacean. In this research, experiments of artificially induced gynogenesis and acrosomal reaction related gene were carried out to explore these problems.X-ray was used to radiate intravital male shrimps thus can further destroy genetic materials in sperms. Also, trypsinase digestion was taken to acquire free sperms from spermatophore, which can provide preconditions for further study on sperms of freshwater shrimps. Besides, Mn SP gene was cloned from ovary of female shrimps, and q RT-PCR analysis showed that this gene has something related with embryonic development and gender differences, this research lays the foundation of further research on Mn SP gene. 1. Trypsinase Digestion in Acquiring Free Sperms from Macrobrachium nipponensisTryptic digestion was used to acquire free sperms with high quantity from spermatophores of Macrobrachium nipponensis. Viable sperms of spermatophore per gram were identified by normal morphology, intact structure and not being colored by trypan blue, the optimum enzyme processing conditions were determined according to the results of sperm morphology observing and trypan blue dyeing. Under the condition of p H 7.4 and different reaction temperature(30℃, 35℃, 40℃ and 45℃, respectively), trypsinase concentration(0.2%, 0.4%, 0.6% and 0.8%, respectively) and reaction time(5min, 10 min, 15 min and 20 min, respectively), the best reaction condition of trypsinase digestion method was studied by orthogonal tests, and the sperm amounts got from each gram of spermatophores were used as a criterion. According to the results of orthogonal tests, we found that trypsinase concentration was the primary factor, followed by reaction temperature and enzymatic reaction time. The maximum effect of trypsinase digestion worked at trypsinase concentration 0.8%, time 10 min and temperature 40℃. This method of acquiring free sperms is useful for further study on sperms of Macrobrachiu nipponensis. 2. Primary study of the effect of X-ray on inactivating sperms by radiating intravital Macrobrachium nipponensisThe effect of X-ray on sperm inactivation was investigated in Macrobrachium nipponense. Male shrimps were irradiated by 100 k V-5m A X-ray for 0, 30, 60, 90 and 120 min with a distance of 10 cm, then mating with female soft-shelled shrimps. Broody days and aberration rate were recorded after ovulation. No difference between 0min group and 30 min group was found, and the aberration rate was 0. With increasing irradiation time, broody days were gradually decreased while aberration rate increased during 30-90 min. After 90 min, broody days showed no change, and the aberration rate reached a peak of 100%. According to the results, the optimum irradiation time to inactivate genetic material of sperms of Macrobrachium nipponense is 90-120 min. Comet assay was used to detect the destruction effect of genetic materials by X-ray. According to the results of comet assay, the mechanism of genetic materials destruction by X-ray was presumed to be denaturation of chromosome, which destroyed the construction of chromosome. 3. Serine proteases gene of Macrobrachium nipponense: molecular cloning and m RNA expression analysisSerine proteinase is an important enzyme participating in acrosomal reaction, and plays a role in the progress of sperms’ permeating in eggs. The serine proteases Mn SP gene( Genbank number: KP091755) from the oriental river prawn, Macrobrachium nipponense was cloned using expressed sequence tag(EST) analysis and a rapid amplification of c DNA ends(RACE) approach. The full-length c DNA of Mn SP was 1792 bp, consisting of a 5′ untranslated region of 132 bp, a 3′ untranslated region of 589 bp, and an open reading frame of 1071 bp. The deduced protein has 357 amino acid residues with a molecular mass of 39.51 k D. Sequence comparison analysis between Mn SP and serine proteases genes from other species showed the similarities ranged from 31% to 42%.Quantitative real-time RT-PCR analysis of Mn SP in different tissues: Mn SP gene was with the highest level in gill in both female and male, much higher than that in hepatopancreas(P<0.05), which means Mn SP gene may has something related with breathing as well as immunity. No Mn SP gene level was detected in muscle shows that Mn SP gene has nothing to do with growth of adult Macrobrachium nipponense. The gene level in ovary was significantly higher than that in testis and vas deferens, also in all the same tissues(gill, hepatopancreas and heart) of both male and female, Mn SP gene shows higher expression level in female than that in male. These results show that Mn SP gene has a different existing pattern in male and female.Quantitative real-time RT-PCR analysis of Mn SP during different developmental stages: The results showed that Mn SP gene was existed in all of the developmental stages of Macrobrachium nipponense, and with higher level during embryonic developmental stage than that in other stages. The highest level was detected in blastula stage, as cell differentiation is the most proliferative in this stage, Mn SP gene may be related with differentiation of embryonic cell, and the effect of Mn SP gene on larva development reduced with the development of Macrobrachium nipponense.Tryptic digestion was taken as the first time to acquire free sperms with high quantity from spermatophores of Macrobrachium nipponensis. The best treating condition of digestion was revealed for further study about sperms of Macrobrachium nipponensis. X-ray was used to destroy the genetic materials of Macrobrachium nipponensis, and comet assay was taken to explore the mechanism of the destruction of X-ray on chromosome, which lays the foundation for further study on artificially induced gynogenesis. Mn SP gene was cloned from ovary of Macrobrachium nipponensis, and q RT-PCR was used to detect the gene level in different tissues and in different developmental stages, that provides precondition for further study on this gene. |
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