斑马鱼二价体制备与多重带显带的方法学探讨

采用碱性低渗结合高氯仿一步显带技术获得了斑马鱼二价体高分辨多重G带,所出现的带纹丰富,反差明显。采用地高辛为标记的以限制性内切酶AfuⅠ介导的原位切口平移技术使斑马鱼二价体上呈现出类G带的多重带,获得了明暗反差强烈的带纹结果。通过比较多个不同分裂相的多条染色体,发现带纹的分布具明显特征性．并且特征一致,带纹数目基本吻合。首次从方法学上对斑马鱼二价体的制备过程及多重带显带技术进行了分析、探讨和总结,力求将该技术程序化、系统化,使其具有可重复性和可操作性,并对显带的可能机理进行了讨论。     

用地高辛标记原位杂交技术定位黄鳝rRNA基因于二价染色体3q12—q24和7…

以地高辛标记重组质粒ＰＴＡ７１中所含的小麦ｒＲＮＡ基因作为探针,与ＥｃｏＲＩ酶切的黄鳝核基因组点ＤＮＡ经Ｓｏｕｔｈｅｒｎ杂交,呈现２条带,片段长度分别为１２．８ｋｂ和４．６ｋｂ。再运用染色体原位杂交技术及杂交一多重带显带技术,定位ｒＲＮＡ基因于黄鳝二价染色体３ｑ１２－ｑ２４和７ｑ１４－ｑ２６两个区间,其分布位点与硝酸银染法结果相符。此外,还讨论了在黄鳝二价体上开展基因定位研究的突出优点。     

用地高辛标记原位杂交技术定位黄鳝rRNA基因于二价染色体3q12─q24和7q14─q26

以地高辛标记重组质粒ＰＴＡ７１中所含的小麦ｒＲＮＡ基因作为探针,与ＥｃｏＲＩ酶切的黄鳝核基因组总ＤＮＡ经Ｓｏｕｔｈｅｒｎ杂交,呈现２条带,片段长度分别为１２．８ｋｂ和４．６ｋｂ。再运用染色体原位杂交技术及杂交后多重带显带技术,定位ｒＲＮＡ基因于黄鳝二价染色体３ｑ１２－ｑ２４和７ｑ１４－ｑ２６两个区间,其分布位点与硝酸银染色法结果相符。此外,还讨论了在黄鳝二价体上开展基因定位研究的突出优点。     

家猪减数分裂粗线期二价体与有丝分裂中期染色体的比较研究

以性成熟公猪睾丸和外周血为材料,采用长低渗、高氯仿卡诺固定液固定和外周血细胞培养制备减数分裂粗线期二价体和有丝分裂中期染色体,通过对二价体和有丝分裂中期染色体分裂指数和长度的比较研究,发现二价体的分裂指数和长度分别是有丝分裂中期染色体的5倍和3．42倍（1．87～5．98）;同时以12号染色体为例,比较了二价体上的染色粒结构带与有丝分裂中期染色体G－带,表明染色粒结构带比中期染色体G－带纹丰富,而与早中期G－带带织吻合。     

斑马鱼粗线期二价体高分辨多重带模式图构建

采用碱性低渗结合高氯仿固定法, 由斑马鱼(Danio rerio)初级精母细胞获得了分散良好的减数分裂粗线期二价染色体分裂相, 且二价体上具有高分辨多重带, 其带纹特征和数目相对稳定, 重复性好. 参照人类细胞遗传学的国际命名法体制, 构建了斑马鱼粗线期二价染色体599条带纹的高分辨带模式图, 并对每条二价体进行了区带划分, 描述了其识别要点.     

黄鳝二价体上微量DNaseⅠ敏感性和限制性内切酶原位切割分析

采用DNase Ⅰ超敏感性分析和限制性内切酶介导的原位切口平移技术(Restriction Enzyme Nick Translation,RE-NT)对黄鳝二价体基因组结构进行了分析研究。已知DNaseⅠ超敏感性与潜在活性基因分布密切相关。结果表明,经DNaseⅠ介导的原位切口平移处理,在黄鳍二价体上可展现类D带带型,而由限制性内切酶介导的原位切口平移结果显示,AluⅠ和MspⅠ均在黄鳝二价体上诱导产生类G带带型,HpaⅡ和HaeⅢ则优先切割5号二价体上一特定区域,诱导出一段由标记信号所构成的类C带,对上述结果进行了分析和讨论。     

家猪减数分裂粗线期二价体与有丝分裂中期染色体的比较研究

以性成熟公猪睾丸和外周血为材料,采用长低渗、高氯仿卡诺固定液固定和外周血细胞培养制备减数分裂粗线期二价体和有丝分裂中期染色体,通过对二价体和有丝分裂中期染色体分裂指数和长度的比较研究,发现二价体的分裂指数和长度分别是有丝分裂中期染色体的5倍和3.42倍(1.87～5.98);同时以12号染色体为例, 比较了二价体上的染色粒结构带与有丝分裂中期染色体G-带,表明染色粒结构带比中期染色体G-带带纹丰富,而与早中期G-带带纹吻合。 Abstract:Meiotic pachytene bivalents were obtained from porcine testes using prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution. Mitotic metaphase chromosomes were prepared from blood cell culture. Comparative studies on division index and length of pachytene bivalents and mitosis metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87～5.98) times longer than the latter, respectively. Chromomere maps of bivalents are more abundant than mitotic metaphase G-bands, while they are correspondent with mitotic early-metaphase G-bands. The result was found by using the chromosome 12 as a sample.     

一种联会消失突变水稻的发现及其细胞学研究

从品种轮回４２２的群体中发现一不育单株,经细胞学观察证明是发生了消失突变,由于该植株的花粉母细胞在减数分裂终变期晚期平均有８．６９个二价体发生了消失,占二价体总数的７２．４％,发生联会消失的最多二价体数目为１２,根据联会消失的最多价体数目以及单位细胞的二价体频率,确定该联合消失变异为完全联会消失变异。     

黄鳝二价体上微量DNaseI敏感性和限制性内切酶原位切割分析

采用ＤＮａｓｅＩ超敏感性分析和限制性内切酶介导的原位切口平移技术（ＲｅｓｔｒｉｃｔｉｏｎＥｎｚｙｍｅＮｉｃｋＴｒａｎｓｌａｔｉｏｎ,ＲＥ－ＮＴ）对黄鳝二价体基因组结构进行了分析研究,已知ＤＮａｓｅＩ超敏感性与潜在活性基因分布密切相关,结果表明：经ＤＮａｓｅＩ介导的原位切口平移处理,在黄鳝二价体上可可展现类Ｄ带带型,而由限制性内切酶介导的原位切口平移结果显示,ＡｌｕＩ和ＭｓｐＩ均在黄鳝二价体上诱导产     

阿拉拉特小麦(Triticum araraticum)抗白粉病基因向普通小麦转移 Ⅰ.阿拉拉特小麦与普通小麦杂种后代的细胞遗传研究及抗性鉴定

配制了普通小麦与阿拉拉特小麦的正、反交组合２０个,杂交结实率为４．９％～３３．６％。不同组合杂种Ｆ１每个ＰＭＣ平均的单价体为１５．２０～１８．５５,二价体为７．０３～９．０２,三价体和四价体分别为０．３６～１．１５和０．０１～０．０２。通过对杂种后代连续２年成株期混合菌种抗性鉴定和苗期分小种分菌系鉴定表明,从普通小麦中国春与阿拉拉特小麦的杂种Ｆ３和Ｆ４代已选择到对白粉病高抗～免疫的单株,它们具有４２条染色体,在ＰＭＣ′ｓＭＩ形成０．００～０．４６个单价体,２０．７７～２１．００个二价体,０．００～０．０６个四价体,在细胞学上已稳定。与已知白粉病抗性基因比较的抗谱分析表明,阿拉拉特小麦携有主效抗病基因Ｐｍ２,在上述的杂交选择过程中,已通过遗传重组将Ｐｍ２基因导入到中国春中。     

百日草根尖和花药愈伤组织染色体制片技术

以种子萌发根尖和花药愈伤组织为材料,研究了取样时间、预处理方法对百日草染色体制片的影响。结果表明:根尖上午8:00～9:00,花药愈伤继代3～5d上午9:00～10:00为最佳取样预处理时间;采用三种药剂预处理活体根尖,以4℃下饱和对二氯苯溶液或0.002mol/L的8-羟基喹啉液预处理8h效果最佳,花药愈伤则以饱和对二氯苯溶液预处理6h效果最佳。本实验的预处理温度是固定的,可克服预处理随季节和时间温度的变化而带来的不稳定性,且百日草花药愈伤染色体观察为首次报道。     

利用粳稻染色体片段置换系群体检测水稻抗亚铁毒胁迫有关性状QTL

潜育性水稻田广泛分布于中国、斯里兰卡、印度、印度尼西亚、塞拉里昂、利比亚、尼日利亚、哥伦比亚和菲律宾等国,其中我国南方稻区就有近700万公顷低产潜育性水稻田。该类水稻田还原性强,矿质营养失调,尤以Fe^2 过量积累,对水稻生长发育产生不良的逆境胁迫作用。培育抗亚铁毒的水稻品种是简便、经济有效地提高稻谷产量的重要途径之一。该文利用由粳稻品种Asominori与籼稻品种IR24杂交衍生的Asominori染色体片段置换系(Chromosome Segment Substitution Lines,CSSLs)群体为材料,检测与抗亚铁毒胁迫有关性状QTL。共检测到与抗亚铁毒胁迫有关性状QTL14个,各QTL的LOD值为2．72～6．63。其中检测到与抗亚铁毒胁迫直接有关的性状叶片棕色斑点指数QTL3个,分别位于第3、9、11染色体C515～XNpb279、R2638～C1263和G1465～C950之间,对应的贡献率分别为16．45％、11．16％和28．02％;与其他已发表的定位结果比较发现,位于第三染色体C515～XNpb279间控制叶片棕色斑点指数的QTL与水稻功能图谱上控制叶绿素含量的QTL的位置一致;表明在亚铁毒胁迫条件下,水稻在其叶片表面出现棕色斑点,叶片衰老,产生一些叶绿素降解物或衍生物,以提高叶片细胞对亚铁等重金属毒害的耐受力。另外,在第11染色体G1465～C950之间检测到了控制叶片棕色斑点指数、茎干重和根干重QTL1个,为主效QTL。在第6染色体XNpb386～XNpb342之间检测到控制茎干重、株高、根长和根干重QTL1个,是否与水稻抗亚铁毒有关需要进一步研究。本研究旨在通过定位与抗亚铁毒有关的QTL,借助与之紧密连锁的分子标记有效地聚合这些QTL,培育出抗亚铁毒性强的水稻新种质材料。     

NaCl胁迫对大麦细胞分裂及染色体行为的影响

NaCl溶液培养导致大麦幼苗根尖细胞有丝分裂指数下降,细胞姐妹染色单体交换(SCE)频率增高,且诱发包括染色体断片、后期染色体桥、不均等分裂及间期细胞微核等的染色体行为异常。细胞平均SCE频率及异常分裂细胞的比率与NaCl浓度和作用时间呈正相关。结果提示：NaCl浓度高或作用时间较长时对大麦细胞具有遗传学毒性。 Abstract：The effects of NaCl solution on chromosome behavior and sister chromatid exchanges(SCE)of barley were studied.Abnormal chromosome behavior including chromosome fragmentation,micronuclei,anaphase bridges and unequal split was found in root tip cells of Hordeum vulgare seedlings.Mitotic index decreased but SCE frequency increased significantly when barley incubated with NaCl solution.The effects of NaCl solution depended on its concentration and treatment duration.The higher the treated concentration was,the higher the ratio of chromosomal aberration was.The longer the treatment duration was,the higher the degree of the effects was.The results showed that NaCl solution was genotoxic when the concentration was higher and the treated time was longer.     

小麦花培苗染色体加倍技术研究

为了提高冬小麦花培苗染色体加倍效率,分别用不同浓度的秋水仙碱对参试的冬小麦材料的花药愈伤组织、再生植株根系和花培苗分蘖节进行了加倍处理。结果表明,用0.02‰和0.05‰秋水仙碱浓度处理的愈伤组织再生植株结实率达33.3%～61.5%。用0.2％的秋水仙碱浸根处理5 h,结实株率平均高达37.5％。用0.04％的秋水仙碱1%的二甲亚砜溶液浸泡分蘖节的时间应在5～10 h之间较为适宜,结实株率平均可达50％以上。     

Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome

To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.     

Cu^2＋对大蒜生长的影响及大蒜根，叶及蒜瓣对Cu^2＋的累积

研究不同浓度（10^-6-10^-4mol/L)硫酸铜（CuSO4,5H2O)溶液对大蒜（Allium satiuwn L.)根,叶和蒜瓣生影响及其这些器官对Cu^2 的积累能力,研究结果指出：在106-5-10^-4mol/L,Cu的处理下,Cu严重影响大蒜根和叶生长,大蒜具有较强吸收和积累Cu^2 的能力,随着Cu^2 处理浓度的增加,大蒜根中的Cu^2 含量递增,大蒜经10^-4mol/L,Cu处理,根部积累了大量的Cu,其含量是对照的52倍,在10^-5和10^-6mol/L Cu处理中,根中Cu的含量分别是对照的13倍和1．4倍,Cu主要积累在极中（10^-5-10^-4mol/L Cu处理）,只有少量的转移到叶子和蒜瓣中。     

Improved in situ hybridization and G-banding by pretreatment with Denhardt''s solution and gelatin-chrome alum

Summary Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity duringin situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline—citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 x SSC for 2 min at 70° C. A³H-labelled, cloned DNA fragment of the highly, repetitive AGM component DNA was hybridized to the chromosomes by incubation at 45° C for 16 h. Treatment with gelatinchrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 x Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin afterin situ hybridization.     

Statistical and image analysis of sister chromatid exchange in maize

The present study reports the use of the fluorescence plus Giemsa (FPG) technique, image analysis and statistical methods to assess the sister chromatid exchanges (SCEs) frequency in maize. Roots derived from germinated maize seeds were treated with BrdU solution and fixed. The slides were prepared by enzymatic cellular dissociation, air-drying technique, stained with Hoechst 33258 fluorochrome, and incubated in salt solution. The chromosomes were irradiated with ultraviolet light and stained with Giemsa solution. The FPG technique associated with digital analysis system was used to measure the length of 597 BrdU-incorporated maize chromosomes and to identify 0.5243 SCE per chromosome. A range from 0 to 4 SCE events were classified and the chi-square test (chi2=1.586, P=0.662) showed a good fit to the hypothesis that the SCEs are independent and random events that follow Poisson distribution. The SCE frequencies in long and short chromosome arms corresponded to a mean value of 0.876 SCE microm(-1). Considering that the maize line used in this study contains 5.78 picogram (pg) DNA (2C value) in interphasic G0/G1 nuclei or 11.56 pg DNA (4C value) in metaphase, and that the DNA mean value corresponds to 0.578 pg/metaphasic chromosome, the analysis suggests an occurrence of approximately 0.9 SCE/pg DNA.     

Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies

Calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method to assess structural and numerical chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length chromosomes would decrease the detection sensitivity of numerical and structural chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60) Co-γ rays; ～0.6 Gy/min; 0-30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G(2) /M-PCC cells with extended length chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G(2) /M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0-20 Gy, respectively. The G(2) /M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable chromosome morphology necessary to assess structural chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.     

Study on the Chromosome N-Banding in Plants

The present paper is dealing with the investigation of the chromosome N-banding technique and the N-banding patterns in Hordeum vulgare, Triticum aestivum, Secale cereale, Vicia faba and Allium eepa. Two N-banding techniques were applied. First, the chromosome slides were stained with Giemsa solution. Second, the slides were treated in 1 M NaH2PO4 solution at 92—94 ℃ for 3.5—8.5 min. After rinsing in tap water they were stained with Giemsa solution. The experiments have demonstrated that the N-banding technique is simple and rapid and the banding patterns are distinctive. The data of N-banding patterns indicated that the N bands did not display the nucleolus organisers exclusively. The comparison of the N-banding patterns of these plants with their C-banding patterns shows that in some of these plants although some regions of N-bands and C-bands correspond, there are a number of instances where regions show N-bands but no C-bands and vice-versa. Therefore, a combination of the N-banding and C-banding techniques should be valuable in the cytological identification of plant chromosome. Like the C-bands, the N-bands are also useful markers in cytogenetics.