摘要
研究了利用紫外线诱导栉孔扇贝雄核发育的条件。表明紫外线(254nm)对卵子染色质失活是有效的。在强度为2.8mW·cm-2·s 1的紫外线下照射20s的卵子与正常精子混合后能保持一定的受精率(60.2%),且D形幼虫发生率为0。染色体检查结果显示此时单倍体率最高(49.2%)。说明在强度为2.8mW·cm-2·s-1的紫外线下照射20s是获得雄核发育单倍体的适宜条件。研究发现受精率和D形幼虫发生率随照射时间的增加而下降,遗传失活的卵子与正常精子受精后其胚胎发育至D形幼虫前期停止。实验中各处理组均出现非整倍体。出现的原因可能由于紫外线照射剂量对卵子染色体遗传失活的作用程度不同以及DNA的光修复。
Androgenesis is defined as all-paternal inheritance. Viable androgenetic diploids can be generated by inhibiting the first cleavage to double paternally derived chromosomes,after fertilization of genetically inactivated eggs with normal sperm. It is a technique that could facilitate the production of completely homozygous isogenic lines, examine sex determination, make genetic analysis, and protect endangered species. Gamma and X-rays inactivation are the usual methods used to inactivate egg nuclei. However, since special facilities are required to manage radioactivity safety, these methods are not practical for routine induction of androgenesis. In many fish species, efficient procedures of genetically inactivating eggs and restoring diploidy have been studied and achievements of successful diploid androgenesis were reported. In contrast, in mollusks, there were only a few reports of cytobiological studies on one species of spontaneous androgenetic Corbicula leana, artificial induction of androgenesis has rarely been studied.Chlamys farreri is a main cultured mollusk species in China, as well as an important object of study in mollusk breeding science. In this study, optimum conditions of ultraviolet (UV) ray irradiation for genetic inactivation of eggs were examined to develop simple and safe techniques to induce haploid androgenesis in C. farreri. Mature cultured C. farreri were collected locally in late March and early May from the coast of Weihai, Shandong. Eggs and sperm were obtained by artificially inducing spawning with the stimulations of dryness and raising water temperature from 16 to 20°C. Discharged eggs were collected by suction and rinsed in filtered seawater several times. Suspensions of sperm and egg were prepared at concentrations of 1.0×10~6 sperm per mL and 1.0×10~4 egg per mL by dilution with filtered seawater. Egg suspension (4 mL) was spread on a plastic Petri dish and treated with the UV rays (254 nm) at intensity of 2.8 mW·cm^(-2)·s^(-1) for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, or 70 s. On the completion of irradiation, egg suspension was mixed with normal sperm (0.5 mL) and transferred to a baker for culture at temperature of 19-20℃. During the culture, the fertilization and development rates of each group were recorded and samples of the larvae (trochophores) were collected to determine the ploidy of embryos.The result showed that the ultraviolet ray was effective for inactivating egg chromosomes. The fertilization rate was relatively high (60.2%) and the developmental rate became zero when eggs were irradiated for 20 s at UV intensity of 2.8 mW·cm^(-2)·s^(-1) and mixed with normal sperm. The results of chromosome observation showed that under this condition the rate of haploid was the highest (49.2%). This result indicated that irradiation for 20 s at UV intensity of 2.8 mW·cm^(-2)·s^(-1) was the optimum dose to achieve haploid androgenesis in this kind of scallop. The fertilization rate decreased with increasing irradiation time. This is probably because with the increasing of irradiation dose, the degree of damage of some important factors that control the fertilization potency of eggs and the development of embryos increased. It was also observed that the development of D-shaped larvae decreased with increasing irradiation time, and the development of the genetically inactivated eggs fertilized with the normal sperm terminated before reaching the D-shaped stage. Aneuploids were found in this study. Because ultraviolet irradiation is known to cause pyrimidine dimerization in the DNA helix, which prevents the replication of genome, the occurrence of these aneuploids is probably attributed to different degrees of maternal chromosomal inactivation by UV irradiation and/or DNA repair.
出处
《水产学报》
CAS
CSCD
北大核心
2004年第2期133-138,共6页
Journal of Fisheries of China
基金
国家自然科学基金资助(30170735)
教育部留学回国人员科研启动基金
关键词
栉孔扇贝
雄核发育
紫外线诱导
遗传失活
单倍体
Chlamys farreri
ultraviolet irradiation
androgenesis
genetic inactivation
