【作者】 许建和；

【导师】 张培军；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2005， 博士

【摘要】 本文以牙鲆Paralichthys olivaceus为研究对象,进行了雌核发育牙鲆子二代雌核发育诱导和正常牙鲆同质雌核发育诱导研究;同时在分析福建和浙江大黄鱼Pseudoscieana crocea养殖群体生化遗传结构的基础上,进行了其三倍体诱导及其相关倍性效应研究。主要结果如下:对牙鲆精子进行紫外照射,灭活其遗传物质,在一定照射时间范围(0-180s,剂量0-3770ergs/mm2)内,精子超微结构、存活率和活力无太大变化,但长时间照射(240s,剂量约为4660ergs/mm2)后则会造成精子膜相系统、线粒体和尾部鞭毛的结构的损伤以及精子存活率和活力的下降。精子经紫外照射后与正常卵子受精表现出明显的Hertwig效应,综合囊胚期存活率和孵化率以及初孵仔鱼单倍体率,牙鲆精子遗传灭活的最适紫外照射剂量约为3770ergs/mm2(即照射时间为180 s)。将紫外灭活的牙鲆精子与正常牙鲆卵受精,受精后适当时刻采用温度休克法和静水压法都可成功抑制受精卵的第一次有丝分裂,诱导出同质雌核发育二倍体。15±0.2℃培育水温条件下,冷休克法诱导牙鲆同质雌核发育的最适条件为受精后85min,1-2℃水温下处理45 min;静水压法诱导牙鲆同质雌核发育最适处理参数为受精后85min,600 kg/cm2压力下处理6min。最适合条件下,冷休克组同质雌核发育二倍体诱导率小于1%,而静水压组诱导率最高可达13.01%,表明静水压法较冷休克法更适合牙鲆同质雌核发育的诱导。开口前同质雌核发育仔鱼较异质雌核发育仔鱼存活率低,但二者全长生长无显著差异(P<0.05)。将紫外灭活的牙鲆精子与雌核发育牙鲆卵受精,受精后适当时刻采用冷休克法和静水压法可成功抑制雌核发育牙鲆卵第二极体排放,诱导出雌核发育牙鲆子二代雌核发育。15℃±0.2℃培育水温条件下,冷休克法抑制雌核发育牙鲆卵子第二极体排放的最适条件为受精后5min,0-1℃水温下处理45 min;静水压法抑制雌核发育牙鲆卵子第二极体排放的最适条件为受精后5min,480 kg/cm2压力水平处理5min。冷休克法在最适条件下处理大批量雌核发育牙鲆卵后,各组受精后6h胚胎相对存活率、孵化后6h仔鱼相对存活率和畸型率分别为84.34±1.7%、53.32±5.57%、15.42±1.79%;静水压法处理大批量卵后处理组三者值分别为86.32±5.07%、79.14±2.11%、8±1.43%。表明静水压法较冷休克法更适合雌核牙鲆子二代雌核发育的诱导。诱导组开口前仔鱼存活率明显低于相同阶段的对照组,而同阶段仔鱼全长二者间却无显著差异(P<0.05)。在大黄鱼福建和浙江两养殖群体中筛选出12种同工酶(AAT、AK、ACP、CK、EST、GPI、IDH、LDH、MDH、MEP、PGM、SDH),共记录了34个基因座位,以此进行了群体遗传变异分析。各同工酶的表达在大黄鱼养殖群体中具有明显的组织特异性。浙江养殖群体的多态基因座位比例(P)、等位基因平均数(A)和群体平均杂合度(H)分别为29.41%、1.32、0.06534,都明显高于福建养殖群体对应值(20.58%,1.24,0.03366)。其中,福建养殖群体检测到7个多态基因座位(Aat-1、Aat-2、Acp-1、Mdh-3、Mdh-4、Mep-1、Mep-4),浙江群体检测到10个多态基因座位(Aat-1、Aat-2、Acp-1、Gpi-2、Gpi-3、Mdh-3、Mdh-4、Mep-1、Mep-4、Sdh)。大黄鱼养殖群体存在明显的杂合子缺失现象。除浙江养殖群体中的Acp-1、Gpi-2和Gpi-3符合Hardy-Weinberg遗传平衡定律外,其它多态座位都偏离Hardy-Weinberg遗传平衡定律,表明经累代养殖后,大黄鱼养殖群体中已经存在严重的近交现象。两养殖群体的遗传相似性系数(I)为0.9984,遗传距离(D)是0.0016,遗传分化系数Gst为0.0564。Dm为0.007,显示了福建和浙江大黄鱼养殖群体较近的亲缘关系。诱导实验结果表明,冷休克法和静水压法都可成功诱导出大黄鱼三倍体。冷休克法适合诱导条件为20℃培育水温下,授精后3min,在3-4℃海水中处理8-10min;静水压法适合诱导条件为同样培育水温下授精后3min,在静水压450kg/cm2下处理3min。综合比较各诱导组和对照组胚胎存活率,孵化率和初孵仔鱼畸型率,静水压法诱导效率明显高于冷休克法。同样培育水温条件下,诱导组胚胎发育开始时较对照组滞后,随着进一步发育,其滞后程度逐渐减弱最终消失,其中静水压法诱导组发育至多细胞期后即与对照组发育同步,而冷休克法诱导组则到原肠早期后才与对照组发育同步。大黄鱼三倍体诱导组和对照组孵化后120d的鱼种,全长的增长上没有明显差异;体重在90d前无明显差异,但120d时三倍体诱导组鱼种明显大于对照组(p<0.05)。更多还原

【Abstract】 Chromosome manipulations in left-eyed flounder and large yellow crocker were carried out by temperature or hydrostatic pressure shock after fertilization. Allozyme analysis was used to investigate the genetic structure and variation in cultivated stocks of P. crocea. The main results were shown as follows:Under the intensity of 3770ergs/mm2, UV light irradiation had slight effects on the ultra-structure, survival and mobility of sperm, while over this intensity, UV light irradiation would cause the damage of sperm ultra-structure and mobility. Therefore the optimal UV dosage for genetic inactivation of sperm of P olivaceus was determined as 3770ergs/mm2 in terms of survival, haploid percentage after irradiation.The optimal conditions for mitogynogenesis induction in P. olivaceus were tested by varying the treatment timing, intensity and duration of application of pressure or cold shocks. Treatment optima for cold shocks were 1-2℃for 45 min at 85 min after fertilization (AF) and 600 kg/cm2 for 6 min at 85 min AF for pressure shocks (water temperature, 15±0.2℃). Ploidy determination was carried out by chromosome counting, flow-cytometry and larvae morphological observation. Under the optimal treatment, survival rate of mitogynogenetic diploid larvae in pressure treatment group at hatching stage was 13.01% , which was much higher than those in cold shock group (less than 1%), indicating that pressure shock was more effective than cold shock for inhibiting the first cleavage of eggs of P. olivaceus. Survival rates of mitogynogenetic diploid larvae at 1-4d post-hatching (PH) were markedly lower than those of their diploid control and meiogynogenetic counterparts at the same stage even though no significant differences were observed in total length of their larvae between them before first feeding.The optimal conditions for meiogynogenesis induction in the second generation of gynogentic P. olivaceus were carried out by changing the treatment timing, intensity and duration of application of pressure or cold shocks. Treatment optima for cold shocks were 0–1℃for 45 min at 5 min after fertilization (AF) and 480 kg/cm2 for 5 min at 5 min AF for pressure shocks. G2 meiogynogenetic diploids were obtained by fertilizing gynogentic eggs with UV-irradiated homologous normal sperm (UV intensity, 3770 erg/mm2) and pressure or cold shocking eggs as above. Ploidy investigations were also performed on experimental groups by chromosome counting, flow-cytometry and larvae morphological observation. Under the optimal treatment, survival of embryo 12h AF and larvae at 6h PH and deformed hatchling rate in cold shock group were 84.34±1.7%,53.32±5.57%,15.42±1.79%; while those indices in pressure group were 86.32±5.07%,79.14±2.11%,8±1.43%, respectively, which showed that pressure treatment was more effective than cold shock for retention of the second polar body of eggs from gynogenetic P. olivaceus under either small batch or large volume scale and had less harm to the subsequent embryonic development and larval survival. Survival of G2 gynogenic larvae at 1-4d PH induced by either cold or pressure shock was not different significantly, but markedly lower than that of their diploid control at the same stage. No significance (P<0.05) was observed in total length of larvae between G2 meiogynogens and their control before first feeding.Horizontal starch gel electrophoresis of allozymes was used to investigate genetic variation and stock structure of Fujian and Zhejiang cultivated stocks of P. crocea. 34 loci of 12 allozymes and their expression patterns in 4 tissues (eye, muscle, heart and liver) were examined and recorded, respectively. The results showed that allozyme expression patterns in cultivated stocks of P. crocea were tissue- specific. At the 95% confidence level, 7 Polymorphic loci (Aat-1, Aat-2Acp-1, Mdh-3, Mdh-4, Mep-1, Mep-4) in Fujian stock and 10 Polymorphic loci (Aat-1, Aat-2, Acp-1, Gpi-2, Gpi-3, Mdh-3, Mdh-4, Mep-1, Mep-4, Sdh) in Zhejiang stock were detected. The proportion of polymorphic loci (P, 29.41%), average alleles (A, 1.32) and average heterozygosity (H, 0.06534) in Zhejiang stock were higher than those in Fujian stock (P, 20.58%; A, 1.24 and H, 0.03366). The genetic deviation index (d) indicated that a deficit of heterozygosity existed in both stocks and 7 of 10 polymorphic loci except for Acp-1, Gpi-2 and Gpi-2 in Zhejiang were significant depart from Hardy-Weinberg equilibrium by X2-test (p<0.05) stock. This result showed that severe inbreeding might have existed in P. crocea cultivated stocks due to generations of intensive culture without addition of wild broodstock. The genetic similarity (I) and genetic distance (D) between these two stocks were 0.9984 and 0.0016, respectively, indicating a close genetic affinity of two stocks.The optimal conditions for triploid induction in P. crocea were performed by pressure and cold shocks. The treatment optima for cold shock were 3-4℃for 10min at 3min AF or 450kg/cm2 for 3min at 3 min AF for pressure shock. In terms of survival, deformed hatchling and triploidy rates, pressure treatment was more efficient and suitable than cold shock in triploidy induction of P. crocea. Cold and pressure treatments had effects on embryonic development of P. crocea and embryonic development of both shock groups were suppressed and the first cleavage were delayed 27min in cold shock groups while 17 min in pressure shock groups compared to their control. But the difference was decreased with embryonic development and disappeared before early blastula stage in pressure group and early gastrula stage in cold group. Putative triploid group had a similar growth performance compared to their diploid counterparts during 20-90 d after hatching even though diploids were smaller in body weight and total length (differences not significant). But 90 -120d after hatching, weight gain in putative triploid group was significant higher than that in control group while difference in length gain at the same stage was not distinct (P<0.05).更多还原