重复DNA顺序pRRD9在稻属中的拷贝数测定

本文应用狭缝印渍杂交方法,把水稻基因组总DNA和含水稻中度重复顺序片段的质粒(pRRD9)DNA分别转移到尼龙膜上形成狭缝印渍、然后用~(32)P标记的 pRRD9插入片段进行杂交、根据各狭缝印渍的放射性强度,测定水稻(Oryza)一些栽培种和野生种基因组中重复DNA顺序的拷贝数,并就拷贝数与水稻进化关系及基因组型的联系进行讨论.     

水稻(Oryza sativa L.)核基因组存在叶绿体psbA基因的同源片段

序列比较说明,重复DNA顺序pRRD9与水稻叶绿体基因组中编码QB蛋白的psbA基因存在高度的同源。用pRRD9亚克隆片段pRRD9R和片段pRRD9L对水稻的叶绿体和核DNA进行Southern杂交分析,揭示了psbA基因同源片段在某个进化时期由叶绿体基因组转移到水稻核基因组,而且两者在水稻进化过程中的变异程度存在明显的差异。利用它们对野生稻和栽培稻总DNA的Southern杂交分析,显示亚洲栽培稻与AA基因组型的野生稻有较近的亲缘关系,以及在部分野生稻产生特异的杂交带谱,说明它可以作为一种分子探针来研究水稻的进化问题。     

水稻重复DNA顺序克隆pRRD3的研究

用ＤＮＡ复性动力学方法克隆到一个水稻（ＯｒｙｚａｓａｔｉｖａＬ．）中度重复ＤＮＡ顺序。不同限制性内切酶消化和Ｓｏｕｔｈｅｒｎ杂交分析显示,这段重复ＤＮＡ顺序以串联加散布的形式存在于水稻基因组中;序列分析表明在它内部含有一个典型的植物启动子序列ＴＧＴＡＴＡＡＡＴＡ;以ｐＲＲＤ３克隆片段作探针,对水稻３４个品种进行拷贝数测定,在野生稻与栽培稻、籼稻与粳稻之间均存在拷贝数上的明显差异;对ＡＡ基因组不同亚型水稻ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析,得到基因组亚型特异的杂交带谱,说明该重复顺序是研究水稻进化和分类的一个有用探针。     

水稻线粒体DNA酶切带型研究

水稻IR36线粒体DNA经6种限制酶酶切,用脉冲电泳和长距离琼脂糖凝胶电泳分离酶切片段,获得高分辨率的清晰带型。每组酶切片段加和测得水稻IR36线粒体基因组大小分别为227kb(HindⅢ)、253kb(EcoRⅠ)、253kb(XhoⅠ)、294kb(BamHⅠ)、239kb(SalⅠ)和283kb(xbal)采用9个来自水稻和玉米线粒体基因组的基因探针与酶切条带杂交发现,水稻线粒体基因组含有包括编码基因在内的重复顺序。     

克隆片段DNA用于测定Epstein-Barr病毒基因组拷贝数

本文报道用非标记的克隆 EBV DNA 限制性酶解片段作标准,同时以标记的克隆片段R 作探针,经 DNA 点杂交检测每个 H_(18)细胞中 EBV 基因组拷贝数。由于实验中结合用 W 片段作标准 DNA 和探针,测定每个 Raji 细胞中所具有稳定的 EBV 基因组拷贝数,以及每个 Raji 细胞中 EBV 基因组的重复序列 W 片段数均与前人报告的结果基本一致,所以说明了本文所用方法及结果的可靠性。此项技术可在病毒阳性细胞培养物中定量测定病毒核酸,也可用于临床分子病毒学作为常规的核酸杂交技术。     

水稻、高粱、高粱稻重复顺序DNA复性动力学分析

应用DNA复性动力学方法对水稻、高粱以及它们的杂交后代高粱稻的重复顺序DNA进行了分析。配合应用计算机技术,研究了它们基因组结构的概况。发现与母本水稻相比,远缘杂交后代高粱稻基因组在中度重复顺序部分发生了变化。     

恒河猴高重复顺序DNA的分子克隆

哺乳动物基因组中高重复顺序可用限制性内切酶消化DNA经用电泳分离获得,本文用限制性内切酶Bam HI消化恒河猴DNA分离得到一系列高重复片段。用其中最小片段与质粒pBR 322共价连接,转化到大肠杆菌后得到3个带有重组子的克隆。其中PMBI克隆用Bam HI,Pst I酶解,杂交鉴定,证明此克隆是含有重组的恒河猴高重复片段,此片段大小约为340碱基对。哺乳动物基因组相当繁杂,一般在10~9碱基对(b.p)以上,应用复性动力学方法可将其分成高重复顺序、中重复顺序及单拷贝顺序。高重复顺序在基因组中重复频率很高。可达百万次。它有许多家族,这些家族核苷酸顺序相近,但不相同,目前有证据表明,高重复顺序与基因表达及调控有关,与生物进化有关,而且还与DNA的复制及调控有关,因此具有重要的生理功用。为了得到纯的单一高重复顺序,我们应用分子克隆技术,建立了恒河猴高重复顺序DNA的分子克隆。     

一种能表现水稻基因组DNA分化特征的重复DNA顺序

用ＤＮＡ 复性动力学方法克隆到一个水稻中度重复顺序。Ｓｏｕｔｈｅｒｎ 杂交、限制性内切酶分析及序列分析资料表明,该重复顺序在水稻基因组中具有串联重复和散布状态两种存在方式。以该ＤＮＡ 片段作探针,用Ｓｏｕｔｈｅｒｎ 杂交方法分析了多种野生稻种和栽培稻品种的基因组分化特征。某些限制性内切酶消化过的水稻ＤＮＡ,其图谱呈现出多达４０ 条以上的杂交带,包括强杂交带和弱杂交带两种类型。重复实验结果证明,强杂交带表现为ＢＢＣＣ染色体组型特异而弱带则在栽培稻各品种间显示出丰富的多态性,表明该重复顺序片段在水稻理论研究和育种实践中可能具有重要意义     

水稻BAC在玉米有丝分裂染色体上FISH杂交体系的构建

 以水稻细菌人工染色体(BAC)为探针在玉米有丝分裂的细胞学制片上进行荧光原位杂交(FISH),探讨玉米基因组C_ot DNA对BAC探针重复序列的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化、水稻BAC探针的特异性重复序列的封阻对FISH杂交信号特异性的影响.初步形成了一套以水稻BAC探针在玉米有丝分裂染色体上进行BAC-FISH杂交的优化技术体系.研究结果表明：使用玉米基因组C_ot DNA来封阻水稻BAC探针的重复序列玉米基因组C _ot DNA的C_ot值应小于50,同时还需根据不同探针调整C_ot DNA的C_ot值及与探针的比例;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻BAC探针本身特异的重复序列的封阻对BAC-FISH杂交信号特异性的改善具有较好的效果.     

水稻（Oryza sativa L.）核基因组存在叶绿体psbA基因的同源 …

序列比较说明、重复ＤＮＡ顺序ｐＲＲＤ９＾＊与水稻叶绿体基因组中编码ＱＢ蛋白的ｐｓｂＡ基因存在高度的同源。用ｐＲＢＤ９亚克隆片段ｐＲＲＤ９Ｒ和片段ｐＲＲＤ９Ｌ对水稻的叶绿体和核ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析,揭示了ｐｓｂＡ基因同源片段在某个进化时期由叶绿体基因组转移到水稻核基因组,而且两者在水稻进化过程中的变异程度存在明显的差异。     

Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.     

Extraordinarily polymorphic ribosomal DNA in wild and cultivated rice.

A collection of 481 rice accessions was surveyed for ribosomal DNA (rDNA) intergenic spacer length polymorphism to assess the extent of genetic diversity in Chinese and Asian rice germplasm. The materials included 83 accessions of common wild rice, Oryza rufipogon, 75 of which were from China; 348 entries of cultivated rice (Oryza sativa), representing almost all the rice growing areas in China; and 50 cultivars from South and East Asia. A total of 42 spacer length variants (SLVs) were detected. The size differences between adjacent SLVs in the series were very heterogeneous, ranging from ca. 21 to 311 bp. The 42 SLVs formed 80 different rDNA phenotypic combinations. Wild rice displayed a much greater number of rDNA SLVs than cultivated rice, while cultivated rice showed a larger number of rDNA phenotypes. Indica and japonica groups of O. sativa contained about equal numbers of SLVs, but the SLV distribution was significantly differentiated: indica rice was preferentially associated with longer SLVs and japonica rice with shorter ones. The results may have significant implications regarding the origin and evolution of cultivated rice, as well as the inheritance and molecular evolution of rDNA intergenic spacers in rice. Key words : rDNA, Oryza rufipogon, Oryza sativa, germplasm diversity, evolution.     

Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon

Endogenous, 14 kb double-stranded RNAs (dsRNAs) have been found in two ecospecies of cultivated rice (temperate japonica rice and tropical japonica rice, Oryza sativa L.) and in wild rice (O. rufipogon, an ancestor of O. sativa). A comparison of the nucleotide and deduced amino acid sequences of the core regions of the RNA-dependent RNA polymerase domains found in these three dsRNAs suggested that these dsRNAs probably evolved independently within each host plant from a common ancestor. These dsRNAs were introduced into F1 hybrids by crossing cultivated rice and wild rice. Unusual cytoplasmic inheritance of these dsRNAs was observed in some F1 hybrids; the evolutionarily related dsRNAs were incompatible for each other, and the resident dsRNA of an egg cell from cultivated rice was excluded by the incoming dsRNA of a pollen cell from wild rice. Coexisting dsRNAs in the F1 hybrids segregated away from each other in the F2 plants. However, the total amount of these dsRNAs in the host cells remained constant (ca. 100 copies/cell). The stringent regulation of the dsRNA copy number may be responsible for their unusual inheritance.     

野生稻与栽培稻及种间杂种F1叶表面亚显微结构的研究

用扫描电镜对原产中国的３种野生稻、２个栽培稻品种及栽培稻与野生稻间的杂种Ｆ１,就气孔频度、气孔器乳突、大瘤状乳突及本栓细胞乳突等叶片表面亚显微结构作了比较观察研究。结果显示这些性状具有种的特性,发现种间杂种的亚星微性状与两个亲本 特性及亲缘关系远近有关。     

Evolutionary Genomics of Weedy Rice in the USA

Red rice is an interfertiie, weedy form of cultivated rice (Oryza sativa L.) that competes aggressively with the cropin the southern US, reducing yields and contaminating harvests. No wild Oryza species occur In North America andthe weed has been proposed to have evolved through multiple mechanisms, including "de-domestication" of UScrop cultivars, accidental introduction of Asian weeds, and hybridization between US crops and Asian wild/weedyOryza strains. The phenotype of US red rice ranges from "crop mimics", which share some domestication traitswith the crop, to strains closely resembling Asian wild Oryza species. Assessments of genetic diversity haveindicated that many weed strains are closely related to Asian taxa (including indica and aus rice varieties, whichhave never been cultivated in the US, and the Asian crop progenitor O. rufipogon), whereas others show geneticsimilarity to the tropical japonica varieties cultivated in the southern US. Herein, we review what is known aboutthe evolutionary origins and genetic diversity of US red rice and describe an ongoing research project to furthercharacterize the evolutionary genomics of this aggressive weed.     

Evolutionary Genomics of Weedy Rice in the USA

Red rice Is an Interfertlle, weedy form of cultivated rice （Oryza sativa L.） that competes aggressively with the crop In the southern US, reducing yields and contaminating harvests. No wild Oryza species occur In North America and the weed has been proposed to have evolved through multiple mechanisms, Including ＂de-domestication＂ of US crop cultlvars, accidental introduction of Asian weeds, and hybridization between US crops and Asian wild/weedy Oryza strains. The phenotype of US red rice ranges from ＂crop mimics＂, which share some domestication traits with the crop, to strains closely resembling Asian wild Oryza species. Assessments of genetic diversity have Indicated that many weed strains are closely related to Asian taxa （Including indica and aus rice varieties, which have never been cultivated In the US, and the Asian crop progenitor O. ruflpogon）, whereas others show genetic similarity to the tropical Japonica varieties cultivated In the southern US. Herein, we review what Is known about the evolutionary origins and genetic diversity of US red rice and describe an ongoing research project to further characterize the evolutionary genomlcs of this aggressive weed.     

一些水稻的重复DNA顺序在稻属和其它禾本科植物中的多态性

重复DNA顺序是真核生物基因组的特征,很多重复DNA顺序已从小麦、拟南芥菜、燕麦、水稻、玉米等植物的基因组中克隆出来,还发现有一些重复DNA顺序具有基因组特异性,用它们作探针可以分析同属或同科物种的起源和亲缘关系,并建立系统进化树。小卫星DNA或微小卫星DNA所产生的指纹图谱可作为一种遗传学标志来研究系统进化、染色体的精细结构和物种的鉴定。一些中度重复DNA序列还可以作为组织培养株系和细胞杂交筛选的分子标志。稻属已发现并定名的有22个种,根据杂交亲和性、细胞遗传学和生理生化等将它分为6个二倍体组型(AA、BB、CC、DD、EE和FF)和2个四倍体组型(BBCC和CCDD)。现多把禾本科分作5个亚科:竹亚科、稻亚科、早熟禾亚科、画眉草亚科和     

Gene flow from cultivated rice (Oryza sativa) to its weedy and wild relatives

BACKGROUND AND AIMS: Transgene escape through gene flow from genetically modified (GM) crops to their wild relative species may potentially cause environmental biosafety problems. The aim of this study was to assess the extent of gene flow between cultivated rice and two of its close relatives under field conditions. METHODS: Experiments were conducted at two sites in Korea and China to determine gene flow from cultivated rice (Oryza sativa L.) to weedy rice (O. sativa f. spontanea) and common wild rice (O. rufipogon Griff.), respectively, under special field conditions mimicking the natural occurrence of the wild relatives in Asia. Herbicide resistance (bar) and SSR molecular finger printing were used as markers to accurately determine gene flow frequencies from cultivated rice varieties to their wild relatives. KEY RESULTS: Gene flow frequency from cultivated rice was detected as between approx. 0.011 and 0.046 % to weedy rice and between approx. 1.21 and 2.19 % to wild rice under the field conditions. CONCLUSIONS: Gene flow occurs with a noticeable frequency from cultivated rice to its weedy and wild relatives, and this might cause potential ecological consequences. It is recommended that isolation zones should be established with sufficient distances between GM rice varieties and wild rice populations to avoid potential outcrosses. Also, GM rice should not be released when it has inserted genes that can significantly enhance the ecological fitness of weedy rice in regions where weedy rice is already abundant and causing great problems.