rDNA在斑茅染色体的物理定位和染色体基数研究

为了了解5S rDNA和45S rDNA在斑茅染色体上的分布情况及其染色体基数,利用荧光原位杂交技术在斑茅海南92-77(2n=60)和海南92-105(2n=60)的中期染色体和间期细胞核上进行5S rDNA和45S rDNA定位研究。结果表明在海南92-77和海南92-105的中期染色体和间期细胞核上45S rDNA位点均有6个信号位点,且均位于染色体短臂的端部上;这6个45S rDNA位点大小具有明显的差异,说明斑茅的45S rDNA的拷贝数存在差异。5S rDNA位点在海南92-77和海南92-105均有6个信号位点,且均位于染色体长臂的着丝粒附近;5S rDNA位点大小相差无几,说明斑茅的5S rDNA的拷贝数差异不大。因此,可以推断染色体数目为60的斑茅染色体基数为x=10。     

新疆红花随体染色体及25S rDNA位点分析

利用荧光原位杂交技术研究我国新疆红花（Carthamus tinctorius L.）25S rDNA在染色体上的位点数目,同时分析了其随体染色体数目。研究结果显示：新疆红花有2对随体染色体;4对染色体存在25S rDNA位点,其中3对染色体上的杂交信号较强,可稳定检出,有时还可检测到1对染色体上较弱的杂交信号。与国外红花品种相比,新疆红花rDNA位点数目多,表明新疆红花与国外红花品种间核仁组织区结构存在较大差异。这些结果将为研究红花染色体进化及其准确的核型分析提供一定的参考。     

烟草染色体显带技术试验研究

染色体显带技术在烟草遗传育种方面有着重要的应用价值。本试验在比较常规制片和Ｆ一ＢＳＧ酶解火焰干燥制片方法的基础上，提出用６％ＮａＨＣＯ＿３代替Ｆ一ＢＳＧ中的５％Ｂａ（ＯＨ）＿２对染色体进行变性处理，处理时间－２０～２５分钟显带效果较好。用对二氯苯饱和水溶液对烟草根尖预处理３～５小时可以积累较多的细胞有丝分裂相。     

烟草18S rDNA染色体原位杂交技术优化研究

荧光原位杂交（Fluorescence in situ hybridization,FISH）在细胞遗传学中有较多应用,其中重复序列常被作为探针来研究物种进化、分类。但现常用的探针序列均较长（200 bp-500 bp）,杂交耗时较多。本研究以烟草（Nictiana tabacum）为材料,克隆并标记18S rDNA保守序列（254 bp）,以其为探针对烟草染色体进行FISH,经18 h杂交获得了清晰的信号。在此基础上,利用荧光素直接标记引物序列（寡聚核苷酸）,经较短时间（2 h）的杂交,也获得了清晰的信号。以寡聚核苷酸为探针对云烟87四倍体、N.tabacum-N.plumbaginifolia五倍体、烟草六倍体、云烟87八倍体以及N.plumbaginifolia的染色体进行杂交,均获得了良好效果。且杂交液可简化配制,洗脱过程也可简化。18S rDNA寡聚核苷酸探针与5S rDNA寡聚核苷酸探针结合,实现了N.plumbaginifolia的18S rDNA和5S rDNA双色FISH。所以,该寡聚核苷酸序列可以应用于烟草属植物18S rDNA（45S rDNA）位点的分析,且较大程度缩短了操作时间并简化了操作过程。      

烟草单倍体植株减数分裂的研究

本文阐明了烟草单培体花粉植株小孢子母细胞的减数分裂和小孢子发育的过程。发现由于前期Ⅰ时没有同源染色体配对,中期Ⅰ时染色体分散于细胞质中,绝大多数细胞的染色体不排列于赤道板上,仅少数细胞有赤道板的形成。后期Ⅰ时单价染色体随机分布两极,故末期Ⅰ形成大小不等的双核或多核。减数分裂Ⅱ为两条染色单体分离,表现基本正常,形成含有两两相等染色体数目的四核或多核。减数分裂还发现了异常的连续型分裂和四分孢子呈平面体型。小孢子较小,平均直径为16μ,与二倍体烟草的小孢子相比,经测定其差异达到极显著水平。小孢子的细胞质退化,未发现有丝分裂,基本上形成不育的小孢子。     

烟草Hsp70基因家族的鉴定及NtHsp70Chl基因的表达分析

为探明烟草热激蛋白70（Hsp70）基因家族的特征及烟叶主脉中NtHsp70Chl基因在烘烤条件下的表达模式,对Hsp70基因家族进行了亚细胞定位、系统进化、基因结构、染色体定位,并对NtHsp70Chl基因进行了烘烤条件下实时荧光定量表达PCR分析。结果表明,烟草基因组中Hsp70基因家族共有61个成员,蛋白序列长度不等,等电点在4.52~9.75范围内,各成员蛋白分别定位于细胞质、内质网、细胞外基质、叶绿体和线粒体中;烟草Hsp70基因家族分为6组,定位于叶绿体的成员均存在Ⅵ-a亚组;61个NtHsp70基因分布于18条染色体上,经亚细胞定位于叶绿体的基因分别位于6、17和19号染色体上;烟草Hsp70家族中具有10个基因重复和5个旁系同源基因;经RT-qPCR检测分析,定位于烟草叶绿体中的NtHsp70Chl基因对高温诱导的敏感性较弱。本研究为深入研究烟草Hsp70的功能奠定了基础。     

以子房为材料制备烟草染色体标本的方法

为使烟草染色体标本制备过程中取材更方便,本研究以异源五倍体烟草（2n=5x=58）为材料,观察了不同大小子房、不同预处理方法对烟草染色体制片效果的影响。首先,子房大小按花冠与花萼长度比例分为6类;其次,预处理方法分为2类,即：（1）室温下于0.002 mol/L的8-羟基喹啉中预处理2、4和6 h;（2）7℃下于0.002 mol/L的8-羟基喹啉中预处理12、24和36 h。结果显示,当花冠长度小于或等于花萼长度的1/2时,子房中分裂期细胞的比例最大,为97.00±17.82个/1000个。该时期子房于7℃经24 h预处理后,染色体数目与形态清晰可辨。利用上述方法制作云烟87（2n=4x=48）和N.plumbaginifolia（2n=2x=20）的染色体标本,均获得较好的结果。此外,将上述方法制作的五倍体烟草染色体经5S rDNA-FISH,获得了清晰可辨的5个不同信号,与预期一致。可见,幼嫩子房可用于烟草染色体标本的制作,且用改良的预处理方法制备的染色体标本可用于染色体计数和核型分析,也可用于原位杂交分析。     

基于RAD重测序技术开发烟草品种SNP位点

利用限制性内切酶位点标签（RAD）技术,通过对10份供试烟草材料的基因组简化重测序,发掘了烟草高通量SNP位点,为烟草基因组学提供标记信息。结果表明,本研究共获得了44.33 Gb的Clean data数据,平均覆盖度1.01 X,共鉴定到291 770个SNP位点,SNP位点间的平均间距为10.066±29.801 kb。发掘到的SNP位点能够覆盖整个基因组,但在不同染色体部位上的分布密度存在一定差异,在17号染色上半臂的存在一段大范围的SNP密集区域。SNP变异类型以转换为主,通过功能注释在基因区域发现45 049处SNP位点。利用SNP分型信息,计算了供试品种间的遗传距离,平均为0.29,台烟8号的遗传背景与其他品种相对最远。该结果将为烟草QTL定位、候选基因发掘、亲本组配等研究提供科研依据。     

NtSPX4基因敲除对烟草苗期磷吸收和生长发育的影响研究

[目的]为探究NtSPX4基因在烟草磷吸收转运中的作用.[方法]同源克隆了烟草(Nicotiana tabacum L.)K326中NtSPX4基因,通过CRISPR/Cas9技术获得了NtSPX4-1和NtSPX4-2均发生纯合突变的K326株系S42,在不同磷浓度的Hoagland溶液培养条件下,对S42株系的生长...     

烟草橙蛋白基因的克隆与生物信息学分析

为了获得烟草OR(NtOR)基因的全长序列和进一步揭示其在特色烟叶形成中的作用,采用同源克隆法从烟草品种K326中克隆了NtOR的全长cDNA和基因组DNA序列,GenBank登录号为JN379458和JN375578.生物信息学分析表明,NtOR基因组DNA全长5027bp,cDNA为1188 bp,编码317个氨基酸残基.NtOR蛋白具有2个跨膜域、1个信号肽剪切位点、5个糖基化位点和11个磷酸激酶修饰位点,二级结构包含12个α-螺旋和20个β-折叠,亚细胞定位于质膜上.系统进化与BLAST分析表明,NtOR是SlOR和ViOR的垂直同源基因.GENEVESTIGATOR数据库的芯片结果显示,NtOR基因在子叶中的表达量最高,其次是成熟叶片、幼茎和花,其他组织器官的表达相对较弱.     

水体中烟草花叶病毒的浓缩与检测方法

为了检验水体中TMV的浓缩和检测方法的效果,在含和不含土壤颗粒的水中加入感染烟草花叶病毒(TobaccoMosaicVirus,TMV)病叶汁液,然后用低速离心法和带正电荷微孔滤膜吸附法浓缩,再用接种心叶烟、普通烟K326和间接酶联法检测浓缩物中TMV存在与否。含土壤颗粒的样品的离心法浓缩物用接种心叶烟检测,同一个稀释度离心浓缩比不处理对照的枯斑数明显要多。说明离心法有一定的浓缩效果。接种K326检测法、离心浓缩法、上清液和不处理(对照)的最低检出稀释度接近,说明离心浓缩方法的回收效果较差。用酶标板间接ELISA检测,离心处理的最低检出稀释度低于离心上清液和不处理对照。推测可能原因是土壤滤出液降低酶标板吸附TMV的效率。含和不含土壤颗粒的样品吸附法浓缩物用接种心叶烟检测,同一个稀释度吸附浓缩比不处理对照的枯斑数明显要多,吸附浓缩的最低检出稀释度比不处理对照高3个稀释度,说明吸附法有一定的浓缩效果。用接种K326检测,吸附浓缩的最低检出稀释度为1:2500倍(土壤滤出液+病叶汁液)和1:5000倍(自来水+病叶汁液)。吸附滤出液检不出TMV。用酶标板间接ELISA检测,吸附处理比吸附滤出液的最低检出稀释度高5~6个梯度、比不处理对照高4个稀释度,同样说明吸附法有较好的浓缩效果和回收效果。吸附法可用于水体病毒浓缩。      

异质性体细胞杂种雄性不育性遗传及其生物学性状研究

普通烟草(Nicotiana tabacum)和粉蓝烟草(N. glauca)的叶肉原生质体融合再生出的体细胞杂种植株,其形态偏普通烟草,雌蕊发育正常,雄蕊发育异常。将融合双亲部分细胞质和细胞核的杂种株与普通烟草栽培品种(Speight G-28♂)回交,回交一代(BC₁)、回交二代(BC₂)保持其雄性不育,回交三代(BC₃)、四代(BC₄)育性有一定的恢复,并出现育性分离。让恢复一定育性株系自交,获得有一定育性的稳定株系,继续与其它普通烟草品种回交(Speight G-80♂,Speight G-140♂,净叶黄♂等),雄性不育性又得到恢复,并稳定地遗传,用普通烟草品种(净叶黄♂)反交时,后代育性恢复正常。对各个世代生物学性状抗病力进行了研究分析。      

Structural characterization of chromosome I size variants from a natural yeast strain

Many yeast strains isolated from the wild show karyotype instability during vegetative growth, with rearrangement rates of up to 10(-2) chromosomal changes per generation. Physical isolation and analysis of several chromosome I size variants of one of these strains revealed that they differed only in their subtelomeric regions, leaving the central 150 Kb unaltered. Fine mapping of these subtelomeric variable regions revealed gross alterations of two very similar loci, FLO1 and FLO9. These loci are located on the right and left arms, respectively, of chromosome I and encompass internal repetitive DNA sequences. Furthermore, some chromosome I variants lacking the FLO1 locus showed evidence of recombination at a DNA region on their right arm that is enriched in repeated sequences, including Ty LTRs. We propose that repetitive sequences in many subtelomeric regions in S. cerevisiae play a key role in karyotype hypervariability. As these regions encode several membrane-associated proteins, subtelomeric plasticity may allow rapid adaptive changes of the yeast strain to specific substrates. This pattern of semi-conservative chromosomal rearrangement may have profound implications, both in terms of evolution of wild strains and for biotechnological processes.     

Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: electrophoretic karyotypes.

Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S. paradoxus and S. bayanus were analysed using contour-clamped homogeneous electric field electrophoresis. Southern blot hybridization with eight cloned S. cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes in the three sibling species. A comparative study of Ty1, Ty2 and telomere-associated Y' sequences having multiple chromosomal location was also done. Chromosome length polymorphism was found in cultured strains of S. cerevisiae. Wild S. cerevisiae and S. paradoxus strains yielded chromosome banding patterns very similar to each other. The karyotype pattern of S. bayanus was readily distinguishable from that of S. cerevisiae and S. paradoxus. Southern blot analysis revealed a low degree of homology between the S. cerevisiae genes studied and the corresponding S. paradoxus and S. bayanus genes. The number of chromosomes appears to be 16 in all three species.     

Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

The PHO80/TUP7 locus in Saccharomyces cerevisiae is on the left arm of chromosome XV: mapping by chromosome engineering.

The PHO80/TUP7 locus in Saccharomyces cerevisiae is reported to be located on the right arm of chromosome XV close to its centromere. In the present study, the locus has been reassigned to the left arm of the same chromosome by reciprocal recombination between chromosomes V and XV at URA3 (on chromosome V) and PHO80/TUP7 loci by using the site-specific recombination system of the yeast plasmid pSR1.     

Extensive chromosome translocation in a clinical isolate showing the distinctive carbohydrate assimilation profile from a candidiasis patient

Variation of the electrophoretic karyotype is common among clinical strains of Candida albicans and chromosome translocation is considered one of the causes of karyotypic variation. Such chromosome translocations may be a mechanism to confer phenotypic diversity on the imperfect fungus C. albicans. A clinical strain, TCH23, from a vaginal candidiasis patient shows distinct carbohydrate assimilation profile, serotype B, no chlamydospore formation and an atypical karyotype (Asakura et al., 1991). To examine the taxonomic relationship among C. albicans, Candida dubliniensis and this strain, we sequenced the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA. The ITS1 sequence of TCH23 was identical with that of C. albicans but not of C. dubliniensis. Thus, strain TCH23 was classified as a variant of C. albicans with an atypical phenotype. The chromosomal DNAs of this strain were resolved into 13 bands on pulse-field gel electrophoresis (PFGE). Using DNA probes located at or near both ends of each chromosome of C. albicans, we investigated the chromosome organization of this strain. Referring to the SfiI map of C. albicans 1006 (Chu et al., 1993), we found that seven chromosomal DNA bands in strain TCH23 were reciprocal chromosome translocations. One homologue from chromosomes 1, 2 and 6 and both homologues from chromosomes 4 and 7 participated in these events. One translocation product was composed of three SfiI fragments, one each from chromosomes 2, 4 and 7. We deduced the breakpoints of chromosome translocation from the physical map of this strain; between 1J and 1J1, between 2A and 2U, both ends of 4F2, between 6C and 6O and both ends of 7F.     

High-Resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; A first step towards an ordered sequencing approach

For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX. A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.