利用RAPD标记鉴定甜菜无融合生殖的同一性

目的：对具有无融合生殖特性的带有白花甜菜染色体的栽培甜菜单体附加系M14三代材料进行同一性鉴定。方法：通过RAPD随机扩增技术对M14三代材料的基因组DNA的扩增多态性进行分析。结果表明,M141998年、1999年、2000年三代材料的RAPD位点高度一致,遗传相似度S＝1,遗传距离D＝0,从而表明M14三代个体完全一样。结论：初步确定M14材料无融合生殖基因在其后代中是稳定传递的。     

无融合生殖甜菜M14的GISH和BAC-FISH研究

无融合生殖是指不经减数分裂和受精作用而产生胚的一种无性繁殖, 因此是胚的克隆, 母系繁殖. 甜菜单体附加系M14是通过二倍体栽培甜菜(Beta vulgaris)和四倍体白花甜菜(B. corolliflora)种间杂交、进而回交, 选育出的具有无融合生殖特性的甜菜品系. 我们利用GISH技术进一步分析了无融合生殖甜菜M14中的染色体情况, 在不封阻的情况下, 附加的外源染色体清晰可见, 这说明栽培甜菜与白花甜菜基因组间种属特异序列的差异明显. 利用无融合生殖甜菜M14和有性生殖栽培甜菜的花期mRNA对白花甜菜第9号染色体的BAC芯片进行了差异杂交分析, 发现2个BAC克隆16-M11, 26-L15含有M14花期特异表达的基因. 选用这2个BAC克隆作为探针, 对具有无融合生殖甜菜M14进行荧光原位杂交, 供试探针均被定位于附加的白花甜菜第9号染色体长臂末端, 呈半合子状态. 本研究BAC芯片的杂交结果结合两种生殖途径中胚和胚乳发育表达方式的保守性可推断, 甜菜中有性生殖和无融合生殖可能共享某些调节因子的相关路径, 正是白花甜菜第9号染色体上的特异基因才使甜菜M14中无融合生殖特性得以表达.     

利用mRNA差异显示技术分离甜菜M14品系特异表达基因的cDNA片段

二倍体栽培甜菜与白花甜菜杂交、进一步回交而获得的单体附加系M14,其染色体组成中除了含有18条栽培甜菜染色体外,还附加有一条野生白花甜菜第9号染色体,该附加染色体通过母本的传递率为96.5%;单体附加系传递率如此高的原因是因为M14中有无融合生殖基因的存在。本实验采用mRNA差异展示技术对甜菜无融合生殖品系M14和正常有性生殖的二倍体栽培甜菜A2Y花蕾减数分裂时期的基因表达进行了差异分析。采用GT15A,GT15G,GT15C3种锚定引物,共筛选了20个随机引物,通过RT-PCR检测,获得了6个阳性差异表达的cDNA片段,应用NCBI的BLASTx软件对测序结果进行同源序列、相似序列检索,为进一步克隆无融合生殖基因提供侯选cDNA片段。     

甜菜单体附加系M14无融合生殖的细胞胚胎学研究

利用常规研究方法,对甜菜单体附加系M 14品系(B eta vu lg aris L.,VV 1C、2n=18 1)的生殖方式进行细胞学与胚胎学研究.结果表明:(1)甜菜单体附加系M 14的4代细胞学检查表明:染色体组分别为VV 1C、2n=18 1;VV 0、2n=18 0;VV 2C、2n=18 2;VVV 0、2n=27 0;VVV 1C、2n=27 1;VVV 2C、2n=27 2等,其中VV 1C、2n=18 1的植株传递率平均为96.7%,表现为稳定传递,具有二倍体孢子无融合生殖特性;其余各种分离植株的传递率总计为3.25%,有性生殖发生率较低.(2)胚胎学研究表明,二倍体孢子无融合生殖的胚珠中,珠孔处看不到花粉管,胚囊没有发生受精作用.2个助细胞提前退化,半数卵细胞的极性与正常卵细胞相反;卵与次生核不经受精而自发分裂,卵细胞自发分裂产生无性胚,次生核自发分裂产生核型胚乳,而且次生核自发分裂早于卵细胞分裂;有性生殖胚珠中,珠孔处可见多条花粉管,胚囊里见到精卵融合的图像.表明甜菜单体附加系M 14是以二倍体孢子无融合生殖为主要繁殖方式,有性生殖为次要敏殖方式的兼性无融合生殖体.     

甜菜单体附加系M14花蕾cDNA文库的构建

为了克隆甜菜单体附加系M14无融合生殖相关基因 ,采用cDNA文库快速构建法制备了M14花蕾cDNA文库。根据甜菜单体附加系M14无融合生殖的细胞胚胎学研究结果 ,提取甜菜M14花蕾三个关键时期的RNA ,分离纯化mRNA ,以Oligo(dT)为引物 ,在逆转录酶的作用下 ,合成第一链cDNA ,进而合成第二链cDNA。含有EcoRⅠ和NotⅠ粘性末端的双链cDNA在T4DNA连接酶的作用下与载体λZAP臂相连 ,并对连接产物进行体外包装 ,得到噬菌体颗粒 ,即甜菜单体附加系M14花蕾cDNA文库。经大肠杆菌寄主菌株XL1-blueMRF’平板检测 ,三个文库滴度分别为 2 .8× 10 5pfu·mL-1、1.6× 10 5pfu·mL-1和 3.5× 10 5pfu·mL-1,克隆重组率为 83%、78%和 81%。cDNA文库可直接用于目的基因的筛选     

利用AFLP技术分析丹顶鹤的亲缘关系

建立了丹顶鹤(Grus japonensis)AFLP分析体系,经筛选,利用28对选择性扩增引物构建了5对丹顶鹤AFLP亲缘关系分析图谱,共得到1 114个扩增条带,其中多态性条带551条,多态性比例为49.5%。每个引物组合扩增的条带数为20～66条,其中,引物E4M1扩增的条带最多,为66条;引物E6M1扩增的条带数最少,为20条。经统计分析,计算了各样品间的相似性系数在0.71～0.88之间,得到5对丹顶鹤的遗传距离,并构建了UPGMA聚类图,结果1号与2号、3号与4号鹤的亲缘关系较近,其余3对鹤(自然配对)亲缘关系较远。表明丹顶鹤具有识别亲缘关系的行为机制,丰富了丹顶鹤繁殖行为机制的研究内容,并为深入研究建立合理的散养丹顶鹤繁育体系提出了建议。     

带有白花甜菜染色体片段的栽培甜菜易位系的筛选

本文采用斑点杂交方法,对近100%传递的甜菜单体附加系M14(2n=19)发生分离的后代反转系(2n=18)进行研究,采用随机引物法标记白花甜菜特异探针,对80株反转系进行杂交实验,结果筛选出19个易位系,这19个易位系有待于进一步研究,证明是否带有无融合生殖基因。     

利用mRNA差异显示技术分离甜菜M14品系特异表达基因的cDNA片段

二倍体栽培甜菜与白花甜菜杂交、进一步回交而获得的单体附加系M₁₄,其染色体组成中除了含有18条栽培甜菜染色体外,还附加有一条野生白花甜菜第9号染色体,该附加染色体通过母本的传递率为96.5%;单体附加系传递率如此高的原因是因为M₁₄中有无融合生殖基因的存在。本实验采用mRNA差异展示技术对甜菜无融合生殖品系M₁₄和正常有性生殖的二倍体栽培甜菜A_2Y花蕾减数分裂时期的基因表达进行了差异分析。采用GT₁₅A,GT₁₅G,GT₁₅C 3种锚定引物,共筛选了20个随机引物,通过RT-PCR检测,获得了6个阳性差异表达的cDNA片段,应用NCBI的BLASTx软件对测序结果进行同源序列、相似序列检索,为进一步克隆无融合生殖基因提供侯选cDNA片段。     

禾本科植物无融合生殖(综述)

禾本科植物包含了世界上最重要的农作物,也包含了最多的无融合生殖的种类,通过无融合生殖可将农作物的F1代杂种优势固定下来,这在固定农作物杂种优势的利用上具有巨大的潜力,然而禾本科植物无融合生殖作为其繁殖多样性的一种形式,在系统进化过程中的作用是非常复杂的,本文统计了禾本科无融合生殖的分布,概述了其无融合生殖的细胞学,遗传学和分子生物学研究进展。     

甜菜无融合生殖单体附加系M14成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14（Betavulgaris,2n=18＋1）为实验材料,利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明：M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态：（1）极性正常的卵细胞,细胞核位于合点端,细胞质含有大量核糖体、线粒体、内质网等细胞器;（2）细胞核位于细胞中央;（3）细胞核位于珠孔端,且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近;未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显,细胞器的种类与数量多,呈现旺盛代谢活动特征,成为二倍体孢子无融合生殖过程中,卵细胞与次生核自发分裂的细胞学标志。     

GISH and BAC-FISH study of apomictic Beta M14

Apomixis is a means of asexual reproduction by which plants produce embryos without fertilization and meiosis, therefore the embryo is of clonal and maternal origin. Interspecific hybrids between diploid B. vulgaris (2n=2x=18) and tetraploid B. corolliflora (2n=4x=36) were established, and then backcrossed with B. vulgaris. Among their offspring, monosomic addition line M14 (2n=2x=18+1) was selected because of the apomictic phenotype. We documented chromosome transmission from B. corolliflora into M14 by using genomic in situ hybridization (GISH). Suppression of cross-hybridization by blocking DNA was not necessary, indicating that the investigated Beta genome contains sufficient species-specific DNA, thus enabling the determination of genomic composition of the hybrids. We analyzed BAC microarrays of B. corolliflora chromosome 9 by using fluorescence-specific mRNA of B.vulgaris and Beta M14. BAC clones 16-M11 and 26-L15 were detected as fluorescence-specifics of BAC DNA of Beta M14. Then both BAC clones 16-M11 and 26-L15 were in situ hybridized to M14 chromosomes. The two hybridized BAC clones were located close to the telomere region of the long arm of a single chromosome 9, and showed hemizygosity. The results of BAC microarrays showed that these developments of embryo and endosperm have conservative expression patterns, indicating that sexual reproduction and apomixis have an interrelated pathway with common regulatory components and that the induction of a modified sexual reproduction program may enable the manifestation of apomixis in Beta species. It would be sufficient for the expression of apomixes with those apomictic-specific genes on chromosome 9 of B. corolliflora.     

GISH and BAC－FISH study of apomintic Beta M14

Apomixis is a means of asexual reproduction by which plants produce embryos without fertilization and meiosis,therefore the embryo is of clonal and maternal origin.Interspecific hybrids between dip-loid B.vulgaris(2n=2x=18)and tetraploid B.corolliflora(2n=4x=36)were established,and then back-crossed with B.vulgaris.Among their offspring,monosomic addition line M14(2n=2x=18 1)was se-lected because of the apomictic phenotype.We documented chromosome transmission from B.corol-liflora into M14 by using genomic in situ hybridization(GISH).Suppression of cross-hybridization by blocking DNA was not necessary,indicating that the investigated Beta genome contains sufficient species-specific DNA,thus enabling the determination of genomic composition of the hybrids.We analyzed BAC microarrays of B.corolliflora chromosome 9 by using fluorescence-specific mRNA of B.vulgaris and Beta M14.BAC clones 16-M11 and 26-L15 were detected as fluorescence-specifics of BAC DNA of Beta M14.Then both BAC clones 16-M11 and 26-L15 were in situ hybridized to M14 chromo-somes.The two hybridized BAC clones were located close to the telomere region of the long arm of a single chromosome 9,and showed hemizygosity.The results of BAC microarrays showed that these developments of embryo and endosperm have conservative expression patterns,indicating that sexual reproduction and apomixis have an interrelated pathway with common regulatory components and that the induction of a modified sexual reproduction program may enable the manifestation of apomixis in Beta species.It would be sufficient for the expression of apomixes with those apomictic-specific genes on chromosome 9 of B.corolliflora.     

Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.     

Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase

The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.     

Variability of cathodic peroxidases in sugar beet (Beta vulgaris L.) cultivars

Eighteen varieties of sugar beet (Beta vulgaris L.), originating from various European countries, were compared in respect of peroxidase variability level. They were cultivated in the same experimental plot. The cultivars differed in ploidy level: one variety was tetraploid, three were diploid and 14 varieties were triploid. The cathodic peroxidase system is controlled by four independent genes, of which only one is polymorphic. Consequently, the varieties were characterised by frequencies of 3 allozymes belonging to one locus. Only one variety proved to be fully monomorphic. Genetic similarities between the cultivars were illustrated by a dendrogram (UPGMA) and show different groups of varieties not related to their ploidy level.     

Genotypic characterization of Salmonella typhi by amplified fragment length polymorphism fingerprinting provides increased discrimination as compared to pulsed-field gel electrophoresis and ribotyping

Amplified fragment length polymorphism (AFLP) is a recently developed, PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In the present study, AFLP was evaluated for its usefulness in the molecular typing of Salmonella typhi in comparison to ribotyping and pulsed-field gel electrophoresis (PFGE). Six S. typhi isolates from diverse geographic areas (Malaysia, Indonesia, India, Chile, Papua New Guinea and Switzerland) gave unique, heterogeneous profiles when typed by AFLP, a result which was consistent with ribotyping and PFGE analysis. In a further study of selected S. typhi isolates from Papua New Guinea which caused fatal and non-fatal disease previously shown to be clonally related by PFGE, AFLP discriminated between these isolates but did not indicate a linkage between genotype with virulence. We conclude that AFLP (discriminatory index=0.88) has a higher discriminatory power for strain differentiation among S. typhi than ribotyping (DI=0.63) and PFGE (DI=0.74).     

Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis

Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.     

Purification and characterization of Tora-bean (Phaseolus vulgaris) lectin

A lectin purified from the Tora-bean (Phaseolus vulgaris) by affinity chromatography with Con-A Sepharose was shown to be a glycoprotein containing 7.8% neutral sugars (D-mannose, N-acetyl-D-glucosamine, L-fucose, and D-xylose, in a molar ratio of 9.6 : 2.0 : 0.6 : 0.7). Its molecular weight was 130,000, as estimated by exclusion gel chromatography, and SDS gel electrophoresis showed that it consists of four subunits of molecular weight 32,000. The lectin reacts with various glycoproteins, i.e., blood group substances, human parotid salivary glycoprotein, fetuin, and bovine submaxillary mucin. Divalent cations, such as Ca2+, Mn2+, and Mg2+, appear to stimulate its reactivity. Inhibition tests using the glycopeptide fragment from fetuin and some oligosaccharides, as well as the binding test with 14C-N-acetyl-lactosamine suggest that the sequence of D-galactose, N-acetyl-D-glucosamine, and D-mannose residues in the carbohydrate chain of fetuin is essential for binding.     

Phylogeny of ten species of the genus Hordeum L. as revealed by AFLP markers and seed storage protein electrophoresis

The phylogenetic relationships of 60 accessions representing ten species of the genus Hordeum were investigated based on AFLP markers and seed storage protein SDS-PAGE electrophoresis. A total of 339 AFLP polymorphic markers were scored as a result of fingerprinting the studied taxa using seven AFLP primer combinations, whereas 46 polymorphic protein bands resulted from the water soluble and water non-soluble seed storage protein electrophoresis. The phylogenetic tree deduced from AFLP analysis is concordant in a large extent with that deduced from seed storage protein electrophoresis. The studied taxa were clustered according to their genome type into two main groups representing the Old and New World’s species. Inside each group the species were clustered according to their genome type. Highly significant cophenetic correlation coefficient was obtained between both AFLP (0.96) and seed storage protein (0.89) indicating the reliability of the results.