【作者】 余舜武；

【导师】 张端品；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2002， 博士

【摘要】 世界最主要的粮食作物都集中在禾本科作物上，如小麦，玉米和水稻。这些作物经长期的人工栽培和少数基因型品种的大面积推广，使这些农作物遗传资源越来越狭窄，受病虫和非生物逆境的危害越来越严重。为了解决世界人口的不断膨胀对粮食需求日益增大的问题，人们正在不断地挖掘野生物种的优良基因资源，不断地通过直向遗传方法将已克隆的基因在其它物种中进行利用。本研究为了了解禾本科作物中两个抗盐相关基因的比较遗传学特点，小麦和水稻与其野生种的杂种遗传学特点，以便充分利用抗盐相关基因和小麦与水稻的野生物种的优良种质资源，探索这些优异资源的使用方法，采用了荧光原位杂交（FISH）和其它遗传学方法进行了研究。 1．利用RHL cDNA克隆作探针，分别在水稻、玉米和大麦染色体上进行荧光原位杂交发现：RHL基因位于水稻第9染色体长臂上；在玉米中检测出2个位点，分别位于第2染色体长臂的近末端和第7染色体长臂近着丝粒处；在大麦中定位于第5染色体长臂末端。按照Gale和Devos 1998年发表在Science杂志中禾本科基因组环形图，发现被定位的玉米和大麦染色体均包含有水稻第9染色体的同源区，基本符合3个物种之间的基因秩序的共线性。但由于基因组大小不同、基因密度不一致以及微共线性保守性程度低，其在各自染色体位置上存在偏差。为了进一步证实RHL基因在玉米和大麦中的存在，通过Touchdown PCR方法扩增出长度一致为518bp的带。用生物素标记的荧光原位杂交技术，以豇豆（Vigna aconitfolilia）中克隆到的P5CS基因作探针对水稻进行物理作图。该探针大约位于水稻第9染色体的短臂近端点处，信号点距着丝粒的相对距离为51.79±1.24％。 2．栽培稻（Oryza sativa，AA，2n=2X=24）和小粒野生稻（O. minuta,BBCC,2n=4X=48）属间有性杂种以及栽培稻和疣粒野生稻（O.meyeriana,GG,2n=2X=24）属间有性杂种由陈葆棠博士提供，两个杂种通过胚抢救获得。通过形态学观察、染色体计数证实了栽培稻和小粒野生稻的杂种状态，进一步应用GISH通过不同的颜色明确地显示了F₁杂种中栽培稻和小粒野生稻来源的染色体。RAPD带型也清晰地揭示杂种中具有双亲的带型。稻瘟病接种实验发现杂种获得了小粒野生稻的部分抗性特点，其感病特点介于双亲之间。栽培稻和疣粒野生稻杂种染色体计数发现数目变化很大，分为两种情况，一种是24条，另一种是28～47条，后一种杂种植株占绝大部分。有丝分裂过程中，染色体先加倍成为四倍体，然后基因组部分分离可以解释该杂种混倍体形成的机制，整个过程类似于减数分裂第一次分裂。GISH结果显示能用两种颜色将杂种中的亲本染色体区分开来，丢失的染色体多半为栽培稻染色体，表明庆粒野生稻在杂种细胞中为优势细胞种。RAPD结果也显示杂种之间的带型差异很大，主要有两种情况，与染色体计数和GISH结果相吻合。结合GISH和RAPD带型统计，与栽培稻的亲缘关系而言，疵粒野生稻（GG）是一种比小粒野生稻（BBCC）更远缘的物种。 3.多花黑麦草作为一种野生牧草，具有许多小麦所不具有的抗病和其它特异优良性状。葛台明博士在1997年通过不对称融合获得小麦和多花黑麦草体细胞杂种。为了使黑麦草的优良险状能最终转移到小麦中，需要用小麦不断地回交以获得回交后代。体细胞杂种生长势差，生根困难，通过不断地使用生根培养基促进生根，然后移栽到大田中，利用不同播期的小麦花粉回交杂种，获得回交后代。通过有丝分裂中期染色体和减数分裂染色体行为观察，发现染色体数目低于预期值，不足42条，减数分裂过程中形成l⁴条单价体，第一次减数分裂后期，有落后染色体出现。为了了解细胞内染色体构成，GISH显示未发现黑麦草染色体的存在。AFLP结果显示体细胞杂种中黑麦草遗传物质相当少，只在少数几个杂种中检测到不超过2%的黑麦草带型，故体细胞杂种为胞质杂种。杂种染色体数目不足42主要是由于组织培养过程中染色体丢失造成的。更多还原

【Abstract】 The most important foodstuff crops in the world concentrate in the grass family (Gramineae), such as wheat, corn and rice. These crops have been cultivated for a long time and only few cultivated varieties have spreaded to great area. The genetic resources of these crops has become more and more limited. Damage from diseases and insects and abiotic stress has become more and more serious. To resolve the problem of global population expansion and great demand for food, fine genie resources from wild species have been continually explored, and cloned orthologous genes have been used widely in other species by transformed methods. To understand genetics characteristics of the two halotolerance genes (RHL and P5CS) of cereal, and the genetic characteristics of hybrids between wheat/rice and its wild species, to utilize the two halotolerance genes and useful genetic resources from wild species of wheat and rice, and to explore the application of fine resources, a serials of genetic research were developed by fluorescent in situ hybridization (FISH) and other genetic methods.1. Through fluorescence in situ hybridization, RHL gene cDNA was mapped on rice, corn and barley chromosomes. RHL gene probe was detected on chromosomes of three species: RHL gene was localized at the interstitial region on the long arm of rice chromosome 9; it has two sites respectively localized at the distal end on the long arm of maize chromosome 2 and near centromere on the long arm of maize chromosome 7; it was also localized at the distal end on the long arm of barley chromosome 5. The result was compared with the grass genomic consensus map published on Science magazine (Gale and Devos, Science, 1998), and the consistency that they all contained homologous region of rice chromosome 6 was confirmed. Further, Touchdown PCR with gene-specific primer also proved the existence of RHL gene with same length in a same region in corn and barley genome. Using a biotin-labeled fluorescent in situ hybridization (FISH) technique, P5CS cDNA clone from mothbean (Vigna aconitifolia) as a probe was approximately located onto the long arm of rice chromosome 9. The percentage distance (from the hybridization sites to centromere) was 51.79 + 1.24%. The prospect of comparative plant biology for crop improvement is discussed.2. Intergenomic hybrid plants between Oryza sativa (AA, 2n=2X=24) and O. minuta (BBCC, 2n=4X=48) and between Oryza sativa and O. meyeriana (GG, 2n=2X=24)were provided by Dr. Chen Baotang, which were produced by embryo rescue after sexual cross. Observation on morphology of hybrid between cultivar rice and O. minuta, chromosome counts confirmed their hybrid status (ABC, 2n=3X=36). Genomic in situ hybridization (GISH) was further applied to establish the parentage of the chromosomes of Fl hybrids, and O. minuta and O. saliva chromosomes were distinguishable in the hybrids by different colors. RAPD profiles unequivocally revealed their hybrids with double parent patterns. The results of blast tests showed that hybrids acquired resistant character from O. minuta, and had a level of susceptibility intermediate between that of the parents; Observation on morphology of hybrid between rice and O. meyeriana, the counts of chromosomes in the hybrids varied in two manners. One is the number of chromosomes was 24. The other is the number of chromosomes was various from 28 to 47, which majoraty of hybrids belong to the latter. Chromosome duplication and subsequent partial separation of the parental genomes during mitosis might explain the results observed in the hybrids between rice and O. meyeriana. The process was similar with the first division of meiosis. The result of GISH revealed the parent chromosomes in the hybrids could be identificated by two colors, but the most of lost chromosomes belonged to the cultivated rice. RAPD profiles suggested the patterns of these hybrids were obviously different. The result of two RAPD patterns accord with the counts of chromosomes and the result of GISH. Combined with RAPD and G更多还原