| Transgenic technology provides an important means for generating organismswith improved traits. The application of microinjection in fish still has manytechnical problems difficult to overcome, while sperm-mediated genetransfer(SMGT) has several advantages to achieve transgenosis in fish. It was foundto be simple, ease of use and able to avoid the limitation of microinjection resultingfrom egg characteristics. However, SMGT technology in fish is at a preliminarystage with problems such as poor reproducibility, SMGT in different fish requirespecific sperm transfer techniques and treatment conditions. Red seabream (Pagrusmajor) and turbot(Scophthalmus maximus), two important marine aquaculturespecies in China, were chosen as research materials in this study, we assessed theeffects of exogenous DNA on the physiological characteristics of their sperm andwhether the exogenous DNA can transfer through the sperm, further explore thefeasibility and improved methods of SMGT technology in this two marine fish. Themain results are as follows:1) The effects of exogenous DNA on the motility of red seabream sperm wasnot significant, the speed of sperm decreased only when the sperm was incubatedwith up to2μg/106cells of pPmvasGFP, the straight line velocity (VSL) was22.0%lower than control group as the concentration of pPmvasGFP up to5μg/106cells,while the curvilinear velocity (VCL) and percentage of motile sperm (MOT) and didnot reduce. Red seabream sperm were treated with plasmid pPmvasGFP andfluorescein-labeled DNA fragments, PCR and fluorescence observation showed thatexogenous DNA adsorbed to surfaces of spermatozoon but not transfer into it.Artificial insemination was performed after sperm treatment with exogenous DNA,no transgene signal was found in red seabream embryos through PCR analyse, the positive tranfection rate was0%, suggesting that the exogenous DNA attached to thesperm were not enough to transfer to F0for producing transgenic red seabream.2) The effects of exogenous DNA on the motility of turbot sperm were notsignificant which was similar with red seabream sperm. Green fluorescence wasobserved in turbot sperm incubated with Fluorescein-DNA, the PCR analysesshowed that plasmid pEGFP-N1cannot transfer into turbot spermatozoon bydirectly incubation, however, positive signal (sequence on pEGFP-N1) was detectedin the DNA extracted from sperm treated with liposome-DNA complexes. DNA wasextracted from embryos after liposome-mediated SMGT, the positive tranfection ratewas0%detected by PCR. Depending on the results we suggested that the exogenousDNA embedded in the sperm cell membrane as it fused with liposome, this part ofexogenous DNA was removed in sperm-egg binding process.This study showed that spermatozoa of red seabream and turbot are not able tospontaneously bind exogenous DNA molecules and deliver it to oocytes atfertilisation. It is difficult to produce transgenic marine fish by SMGT that spermdirectly incubate exogenous DNA. Liposome-DNA complexes can insert exogenousDNA in the sperm cell membrane, which is not sufficient for transgenosis. Theeffects of exogenous DNA on the motility of turbot sperm and red seabream werenot significant. Auxiliary means should be tried to make the exogenous DNApenetrated into the spermatozoon.In addition, this study contains some work of transgenosis. Transcriptionregulation sequences of vasa gene in turbot were cloned by genome walking. Weanalyzed the potential transcript factor binding sites by bioinformatics method,amplified the potential core promoter and linked into plasmid pEGFP-N1,constructed pSmvasGFP expression vector in which green fluorescent protein (GFP)was promoted by transcription regulation sequences of vasa gene of turbot. Furtherresearch will transfer pSmvasGFP into turbot for GFP specific expression inprimordial germ cells (PGCs) of turbot. |
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