人工诱导四倍体大鳞副泥鳅的研究

采用热休克诱导方法研究了抑制第1次卵裂诱发四倍体大鳞副泥鳅的最佳条件。在受精后37min左右，采用40.5℃热水处理4.5min，不但能导致较高的四倍化率，而且胚胎存活率相当高，表明热休克是诱导多倍化的有效方法，需用设备简单，易于推广。     

异源四倍体鲫鲤及其原始亲本的蛋白质研究

用聚丙烯酰胺梯度凝胶垂直平板电泳技术，对异源四倍体鲫鲤及其原始亲本红鲫和野鲤的血清蛋白、血红蛋白和肌肉蛋白进行了比较研究，结果表明，异源四倍体鲫鲤的蛋白质表型主要为原始双亲红鲫和野鲤的共同表达，这3种蛋白质都可以作为异源四倍体鲫鲤的分子遗传标记。     

异源四倍体鲫鲤Sox9a基因保守区序列的克隆及内含子剪接位点分析

用特异于HMG-box保守区的兼并引物扩增了异源四倍体鲫鲤及其原始亲本(红鲫和鲤鱼)的Sox基因. 异源四倍体鲫鲤的扩增产物共有4条带,大小分别约为200,600,900和 1900?bp,而其原始亲本的扩增产物只有3条带,且在雌雄个体中未发现性别特异扩增带. 回收雄性四倍体鱼600?bp扩增带,经亚克隆及测序分析得到一个新的编码71个氨基酸残基的基因. 该基因与虹鳟鱼(Oncorhynchus mykiss)、蟾蜍(Xenopus laevis)、鲤鱼(Cyprinus carpio)等的Sox9基因相似性达97%,据此命名为Atsox9a. 通过计算机分析和RT-PCR方法确定该基因含有内含子,并获得其内含子的序列和剪切位点,此内含子剪切位点在进化上是保守的. Atsox9a对于研究异源四倍体鲫鲤性别决定和分化机制及脊椎动物性别进化具有重要的理论意义.     

人工四倍体鲤鲫的产生

在人工诱导的复合三倍体鲤鱼成熟个体中有极个别个体产出的卵既保留了原有的３套染色体,又能与一套外来精核融合产生四倍体。１９９７年人工产生的８０００多尾鲤鲫四位体鱼经细胞遗传学研究分析鉴定,结果所有的个体都有４套染色体组,染色体数目在１８５￣２１０之间,众数为２００,四位体比率为１００％,这表明人工复合三倍体鲤鱼的生殖方式十分独特,绝大部分个体的成熟卵可借助于外来精子的流动进行自然雌核发育;而有极个别     

小鼠四倍体半克隆胚胎发育研究

半克隆(Semi-Cloned)胚胎是通过注射体细胞核到未去核的卵母细胞中产生的。在半克隆胚胎中,体细胞被用来作为精子的替代物。然而,由于异常的染色体分离,构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响,不能发育到期。本研究通过抑制小鼠半克隆胚胎在激活过程中染色体数目减半,避免非整倍体胚胎形成,研究四倍体半克隆(TetraploidSemi-cloned,TSC)胚胎的发育和体细胞核的掺入对胚胎发育的影响。结果显示,TSC胚胎的体外发育率显著高于二倍体半克隆胚胎,与正常受精卵及孤雌激活对照无显著性差异,但TSC胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少。通过Oct-4染色发现,TSC胚胎囊胚期内细胞团(InnerCellMass,ICM)细胞很少或者没有。移植63个四倍体半克隆胚胎到3只假孕母鼠体内,得到20个胎盘,但没有得到胎儿。组蛋白乙酰化和DNA甲基化检测显示,部分TSC胚胎在囊胚期没有形成正常受精胚胎在ICM和滋养外胚层(Trophectoderm,TE)之间的差异分布。TSC胚胎的基因表达不依赖于细胞分裂次数而依赖于发育时间。虽然TSC胚胎避免了二倍体半克隆胚胎形成非整倍体现象,但由于TSC胚胎没有ICM细胞或ICM细胞很少,所以只能形成胎盘而不能形成胎儿。本实验第一次较为全面地研究了TSC胚胎的发育,同时也为研究体细胞核再程序化、基因打靶技术提供了一种新的途径。     

N+注入对同源四倍体水稻花药组织培养体系的影响

选择三种同源四倍体水稻为实验材料,研究了氮离子注入对其花药诱导愈伤分化成苗组织培养体系影响.结果显示,低剂量范围(1.0×1015~2.0×1015N /cm2)的N 注入能明显提高愈伤诱导率.其可能原因为:低剂量N 注入可使花药组织的膜透性增强,新陈代谢加快,从而使诱导率增加.另外,无论注入剂量大小,N 注入都能明显改善愈伤生长状态,抑制其褐化衰老的进程,还能提高绿苗分化率.对此,从生化角度进行了一定的理论分析.     

四倍体小麦×纤毛鹅观草属间杂种的胚援救及无性系建立

通过活体-离体胚培养在N6无机+5mg/L IBA的培养基上成功地获得了纤毛鹅观草和5个四倍体小麦种(品种)的属间杂种植株及愈伤组织,并建立了杂种植株和胚愈伤组织再生植株无性系. 采用这种培养技术,5个杂交组合的成苗率达12.7%,成愈率达27.4%,有效地克服了纤毛鹅观草和四倍体小麦杂种胚发育小的问题. 对传统胚援救和愈伤组织诱导的方法及远缘杂种中胚发育与亲本形态的关系进行了讨论.     

四倍体湘鲫F3-F4、三倍体湘云鲫、湘云鲤 及有关二倍体的DNA含量

The DNA content of red blood cells of the tetraploid F₃-F₄ hybrids of red crucian carp (♀)× common carp (♂) and their triploid offspring,diploid common carp (Cyprinus carpio),red crucian carp (Carassium auratus red var.),Japanese crucian carp (Carassium auratus cuvieri) and F₂ hybrid was measured by the instrument of the flow cytometer.The DNA content of muscle cells of F₃ hybrid was also measured by the same instrument.The results showed that the DNA content of F₃-F₄ hybrids was about two times of that of the diploid common carp(P＞0.05).The DNA content of the triploid fish produced by mating F₃-F₄ hybrid (♂) respectively with Japanes crucian carp (♀) and Xin-guo red common carp (♀) was about 1.5 times larger than that of the diploid common carp (P＞0.05).F₂ hybrid,Japanese crucian carp,red crucian carp and common carp had the approximate value of the DNA content (P＞0.05).The above results provided further evidence that F₃-F₄ hybrids were tetraploids and F₂ hybrid was diploid.Those data also confirmed that the fish produced by mating F₃-F₄ hybrids respectively with the Japanese crucian carp and Xin-guo red carp were triploids.     

Primary investigation on GISH-NOR in cotton

Six loci of nucleolar organizer region (NOR) were detected in genomic in situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted from Gossypium species as probe. With twice FISH to the same mitotic cell of G herbaceum or G hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs from G hirsutum and Graimondii mitotic images were all terminal types. Four and two GISH-NORs from G herbaceum (var. africanum) were terminal and centromere types, respectively. Six GISH-NORs in G hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image of G raimondii with its own gDNA as probe. From mitotic image of G herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-wholerecovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes in Gherbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.     

化学诱变获得桑树四倍体研究

将０．４％秋水仙素与３％二甲亚砜混合液注入成龄桑生长锥，可诱变产生四倍体枝条。有的桑树品种老条三年累计诱导率达７５％。成龄桑树老条产生的四倍体可提早开花，提早与二倍体杂交培育三倍体。