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题名: 大鳞副泥鳅(Paramisgurnus dabryanus) 和泥鳅(Misgurnus anguillicaudatus)人工雄核发育的研究
作者: 赵振山
答辩日期: 1997
导师: 吴清江
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 泥鳅 ; 大鳞副泥鳅 ; 紫外线 ; 雌核灭活 ; 雄核发育 ; 冷休克 ; 热休克 ; 受精生物学 ; 不同核质关系 ; 同工酶基因表达
摘要: 本文主要包括下列研究内容:1.紫外辐射对泥鳅和大鳞副泥鳅雌核灭活剂量的选择及光复活作用 雌性原核的灭活是鱼类雄核发育育种技术的关键之一。采用紫外线对泥鳅和大鳞副泥鳅卵子雌性原核进行干法灭活以及在人工合成卵巢液、TC-199溶液、Ringer's溶液、Holtfreter's溶液中灭活,并对灭活效果进行了比较。上述四种卵子保存液的用量有10ml(1.57mm深)和20ml(3.14mm深)两种。结果表明,人工合成卵巢液组在90mJ/cm~2的辐射剂量时,可以诱发90%以上的畸形苗,单倍体诱导高峰出现在120-180mJ/cm~2;TC -199溶液、Ringer's溶液组的最佳辐射剂量为120mJ/cm~2; Holtfreter's溶液组的最佳辐射剂量为90-120mJ/cm~2。在上述各辐射剂量下,90%畸形苗率为雄核发育单倍体,而且辐射过程中要不断摇动。灭活效果依次是:人工合成卵巢液 > TC-199溶液 > Ringer's溶液 > Holtfreter's溶液 > 干法,干法灭活效果最差,将泥鳅卵子在人工合成卵巢液中紫外灭活后“受精“,分两组在明暗条件下孵化。当辐射剂量超过120mJ/cm~2时,畸形苗率均达90%以上,经t检验表明,明暗条件下相同辐射剂量组之间均元显著性差异(T < 0.01),光复活作用是极其微弱的。2.雄核发育单倍体胚胎发育以及与正常二倍体组织学比较 雄核发育单倍体胚胎发育表明:从受精后至多细胞期,二倍体与单倍体胚胎发育速度一致,达囊胚期时,单倍体发育开始滞后,在原肠期停留时间较长,胚层下包速度很慢,神经胚期后,各期分期比较困难,其发育速度比正常二倍体慢一个发育期。孵出后单倍体鱼苗具有典型的单倍体综合症。泥鳅卵经紫外辐射灭活雌核后与大鳞副泥鳅精子受精得雄核发育单倍体,大鳞副泥鳅精卵结合得二倍体,在相同条件下(水温24 ℃)孵化,待二倍体发育到鳔-室期(受精后145小时)时,同时取二倍体和单倍体鱼苗做连续切片,比较了两者同时龄鱼苗的眼、心脏、肠、鳃、肾、脑、血液等组织。结果表明:雄核发育单倍体鱼苗的各器官组织存在水肿等实质性病变,而二倍体鱼苗各组织结构发育正常。3.温度休克诱导泥鳅和大鳞副泥鳅雄核发育纯合二倍体 温度休克诱导泥鳅和大鳞副泥鳅雄核发育纯合二倍体的方法是:用紫外线210mJ/cm~2辐射浸泡在人工合成卵巢液中的泥鳅和大鳞副泥鳅卵,使其雌核遗传失活,再与大鳞副泥鳅或泥鳅精子“受精”,在26 ℃下孵化。热休克:“受精”后从15min开始,每隔2min一组将“受精卵”置于39 ℃温水中热休克处理2min,以阻止第一次有丝分裂的发生,结果表明,二倍化诱导率距“受精”后15-19min和27-29min出现两个高峰,大鳞副泥鳅最高二倍化诱导率为61.4%,一批诱导雄核发育二倍体鱼苗达1700余尾,泥鳅最高二倍化诱导率约为13.4%。冷休克:“受精”后13min开始,每隔2min一组将“受精卵”置于7-8 ℃冷水中休克10min, 以阻止第一次有丝分裂的发生,结果表明:在“受精”后15-21min出现二倍化诱导高峰,孵化期的二倍化诱导率最高30.0%,摄食期为14.4%,孵化一周后为5.6%,经染色体和形态特征鉴定表明,冷、热休克诱导的鱼苗为雄核发育纯合二倍体。其染色体数与父本染色体数相同,后代生长状况良好,7个月后经性腺组织切片表明:人工诱导的雄核发育个体性腺发育良好,大多数个体为雌性个体,已有初级卵母细胞形成,雄性个体比较少。4.泥鳅和大鳞副泥鳅雄核发育受精生物学研究 重点观察了紫外失活泥鳅卵子雌核后与大鳞副泥鳅精子“受精”的雄核发育细胞学过程。精子能够正常进入灭活的卵子并形成雄性原核,它的近侧中心粒诱导卵细胞质形成星光体。由于卵核被灭活,观察不到极体排出及雌、雄原核相融合的受精生物学现象,而仅由雄性原核完成细胞发育过程。通过观察雄性原核的第一次有丝分裂变化,结合人工二倍化诱导结果,讨论了细胞各分裂时期的细胞核变化,确定了温度休克诱导雄核发育二倍化的最佳时间发生在S期结果、G_2期早期,次高峰出现在有丝分裂中期。5.不同核质关系下同工酶基因表达与调控的研究 采用聚丙烯酰胺不连续系统凝胶电泳方法,分析了泥鳅和大鳞副泥鳅不同杂交组合、不同核质关系下胚胎发育阶段(0-145小时)中3种同工酶系统(LDH、EST、MDH)的分化表达谱式,所研究的三种同工酶随细胞质、细胞核不同以及胚胎发育各个时期具有不同的同工酶基因表达谱式。以泥鳅卵子为细胞质环境的I、II两组,杂交组(II组)LDH、EST的同工酶基因在受精后3分钟至原肠中期,雄核基因参与表达与调控,部分基因在杂合体中启动与表达的时间比本交组(I组)要早,两组的管家酶(housekeeping enzyme)主要来自细胞质。以大鳞副泥鳅为细胞质环境的III、IV两组,各酶均以细胞质调控为主,其管家酶与泥鳅有很大不同。MDH同工酶在各组合中均未表现出实质性差异,并发现m-MDH可能为特异性酶谱。具体分析和讨论了不同核质关系下同工酶基因的表达和调控的时空顺序,以及雄核发育诱导过程中应考虑的核质关系。
英文摘要: 1. The genetic inactivation of eggs is one of the key techniques in fish androgenesis. This present paper used ultraviolet (UV) rays irradiating for genetic inactivation of eggs. Five methods of irradiation were examined: drymethod and irradiating in a synthetic ovarian fluid (OF), TC-199 solution, Ringer's solution, Holtfreter's solution and the results were compared. The OF and other three solutions were 10ml (1.57mm deep) and 20ml(3.14mm deep) in petri dishes (φ = 9cm) during irradiation. The results showed: when the dosages of irradiation was beyond 90 mJ/cm~2 in OF, more than 90% of fry were deformed and the optimum irradiation dosage were 120 and 180 mJ/cm~2; it was 120 mJ/cm~2 in TC-199 solution and in Ringer's solution; In Holtfreter's solution, the optimum irradiation dosage was 90 and 120mJ/cm~2, More than 90.5% hatching fry were deformed under the optimum irradiation dosages. According to chromosome set analysis, more than 90% of deformed fry were haploids. Moreover, the petri dishes should be stirred during irradiation. The results showed that the order from the best to the worst for inactivation of eggs was OF > TC-199 solution > Ringer's solution > Holtfreter's solution > dry method. The worest method was eggs inactivation with dry method. The eggs of M. anguillicaudatus were irradiated by different UV dosages in OF, then fertilized with sperms of male. The fertilized eggs were separated into two groups and hatched in dark and bright conditions respectively, When UV dosage was beyond 120mJ/cm~2, the deformed fry were more than 90% in both two groups. T-test showed that the deformed rates have not significant difference with the same UV dosages in dark and bright hatching condition (t < 0.01). This means that DNA photorepair of female nucleus is very feeble. 2. The androgenetic haploid embryos development showed that Developmental speed of haploids was similar to that of diploids before poly-cell stage. After early blastula stage, haploids development began to be detained. Gastrula stage stayed so long, blastoderm developing speed was slower than that of diploid. After neural plate stage, it was difficult to distinguish stages, the developing speed of haploid embryos was slower about one stage than that of diploid embryos. Hatched haploid fry had typical haploid syndrome. Androgenetic haploids were obtained by UV irradiating (180mJ/cm~2)M. anguillicaudatus eggs and fertilized with the sperms of P.dabryanus. Diploids were from the fertilized eggs of P. dabryanus(♀) * P. dabryanus(♂). Two kinds fertilized eggs were hatched under the same condition (Temperature:24 ℃), when diploids developed to one-chamber air bladder stage (145h after fertilization), haploid and diploid fry weresampled at the same time and successive sections were taken. By comparison of eyes, hearts, intestines, gills, kidneys, brain and blood of two kinds of fry, the results showed that the organs and tissues of haploid fry exsited substantial pathological changes, such as edemas, but the tissue and the structure of diploid developed normally. 3. The method of inducing diploidization of androgenetic M. anguillicaudatus and P.dabryanus by tempratureshock was that: the eggs of M.anguillicaudatus or P.dabryanus in a synthetic ovarian fluid were irradiated by UV-dosage of 210mJ/cm~2 to assure the inactivation of female nucleus, tenth eggs were fertilized with sperms of P.dabryanus or M.anguillicaudatus and incubated in 26 ℃. Each group of eggs was applied by heat shock at 39 ℃ proceeding 2min to inhibite the first mitotic division of androgenetic haploid at 15min of post-fertilization, each group at intervals of 2min. The results showed that the peaks of androgenetic diploids induction were occured in 15-19min and 27-29min after fertilization. The hightest induced diploid rates of P.dabryanus and M.anguillicaudatus were 51.4% and 13.4% respectively. There were more than 1,700 androgenetic P.dabryanus fry induced by heat shock in one batch. Eggs was applied by cold shock at 7-8 ℃ proceeding 10min to inhibite the first mitotic division of androgenetic haploid at 13min of post-fertilization, each group at intervals of 2min. The results showed that the peak of androgenetic diploids induction was occured in 15-21min after fertilization. The highest yields were 30.0% and 14.4% at hatching stage and feeding time respectively. It was reduced to 5.6% one week after hatching. According to chromosome and morphology of fry analysis, the fry were pure androgenetic diploid, their chromosome numbers and morphology were all-paternal inheritance. The androgenetic diploids grew well. The closer observation on gonad sections of 7 months age individuals showed that gonads of artificial androgenetic diploids developed well, primary oocytes in ovaries had formed. The majority of androgenetic diploids were female, percentages of male were less. 4. The fertilization events of inactivation eggs of M. anguillicaudatus fertilized with the sperms of P. dabryanus were carefully investigated. The results revealed that sperms could enter inactived eggs normally and became male pronucleus and centrioles and induced egg cytoplasm to become aster. Because female genome were eliminated, polar body and zygotic nucleus hadn't be observed, only male pronucleus controlled embryonic development. The changes of male pronucleus during the first mitotic division were mainly observed and discussed. The results showed that the optimum phase of androgenetic diploidization inducing (induced by heat or cold shock) were at end of synthesis phase or the beginning of G_2 phase of cell cycle. The second induction peak were at meta-phase. 5. The expression patterns and regulations of three isozymic systems (LDH, EST, MDH) were investigated in the early developmental stages [from unfertilized eggs to the one chamber air bladder stage (0-145h)] in different combinations between nucleus and cytoplasm of M. anguillicaudatus and P.dabryanus. Isozymes were resoled by polyacrylamide gel electrophoresis and then detected by specific histochemical staining. The expression patterns and regulations of three isozymic systems were differ from the different combinations of cytoplasm and nucleus. Group I、II had the same cytoplasm of M. anguillicaudatus. The male nucleus genes of LDH and EST were expressed in hybridization group (II) from 3min of post-fertilization to mid gastrula stage, however their gene products in inbreeding group (I) were not. Some of the isozymic genes in hybridization group were expressed earlier than that in inbreeding group. The housekeeping enzymes of group I and II were from cytoplasm. Group III and IV had the same cytoplasm of P.dabryanus, the isozymic genes of them were expressed and regulated by cytoplasm, their housekeeping enzymes were very different from that of M.anguillicandatus. The patterns of MDH were the same in all kinds of combination groups. The m-MDH seem to be a spesific isozymic pattern. The temporal and spatial expressions and regulations of isozymic genes in different combinations between nucleus and cytoplam were discussed.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12508
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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大鳞副泥鳅(Paramisgurnus dabryanus) 和泥鳅(Misgurnus anguillicaudatus)人工雄核发育的研究.赵振山[d].中国科学院水生生物研究所,1997.20-25
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