玉米CMS分子生物学研究进展

本文对玉米CMS研究已获得的、并为普遍接受的分子生物学研究结果进行了粗略总结;对近年来在玉米细胞质雄性不育育性相关核基因的分子标记定位、克隆及辅助选择,育性相关胞质基因的克隆与表达方面的研究进展进行了简要概述;我们认为在今后一段时期,玉米CMS研究将着重围绕核不育基因的克隆及表达模式、线粒体功能基因组、育性相关胞质基因与核育性基因相互作用等方向进行研究,以期阐述玉米CMS的形成机理。 Progress of Molecular Biology of CMS in Maize ZHANG Zu-xin,ZHANG Fang-dong,ZHENG Yong-lian National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,China Abstract:In the paper,we have summarized the molecular biological accomplishment acquired and accepted by most of maize researchers on CMS of maize.A brief review of current molecular biological progress of CMS of maize are displayed in the paper.These progresses concern in the positioning,cloning and maker-assisted selection of nucleic genes associated with fertility,expression and cloning of cytoplasmic genes associated with male sterility,In order to elucidate the molecular mechanism of CMS of maize,the areas about cloning and expression profiling of male sterile nucleic genes,and functional genomics of mitochondria,and interaction cytoplasmic genes with nucleic genes will need to be researched in the future. Key words：maize(Zea mays L.);CMS;mtDNA;gene associated with fertility     

与油菜细胞质雄性不育相关的DNA位点和育性恢复基因的研究进展

细胞质雄性不育(cytoplasmic male sterility,CMS)在油菜杂交种子生产中具有重要的意义.文章主要从目前已发现的与油菜CMS相关的线粒体DNA位点,育性恢复基因对CMS相关DNA位点表达的影响,育性恢复基因的分子标记定位和育性恢复基因的克隆4个方面综述了近年来油菜CMS的研究进展.并就该领域今后的研究方向进行了探讨.     

Alterations of RNA Editing for the Mitochondrial ATP9 Gene in a New orf220-type Cytoplasmic Male-sterile Line of Stem Mustard （Brassica juncea var. tumida）

RNA editing for the mitochondrlal ATP9 gene of encoding regions has been observed in both cytoplasmic malesterile and maintainer lines of stem mustard, where its editing capacity varied spatially and temporally in the cytoplasmic male sterility （CMS） line. There were four RNA editing sites for the mitochondrial ATP9 gene according to Its normal editing sites in mustard, of which three sites occurred as C-to-U changes and one as a U-to-C change. As a result, the hydrophobicity of deduced ATP9 protein was reduced due to the conversions at its 17th, 45th and 64th positions. Meanwhile, the conservation of deduced ATP9 protein was enhanced by changes at the 56th position. Loss of a specific editing site for ATP9 was observed in juvenile roots, senile roots, senile leaves and floret buds of the CMS line. Comparatively, complete RNA editing for ATP9 gene was retained in juvenile roots, juvenile leaves and floret buds of its maintainer line; however, the loss of a specific editing site for ATP9 gene occurred at senile roots and senile leaves in its maintainer line. These observations allow us to produce a hypothesis that the dysfunction of a specific mitochondrial gene arising from RNA editing could probably be a factor triggering CMS and organ senescence through unknown cross-talk pathways during development.     

红莲型细胞质雄性不育水稻线粒体DNA的RFLP分析

应用RFLP技术,研究了红莲型不育系、保持系、杂种一代及野败型、马协型不育系的线粒体基因组。结果表明,红莲型不育系与保持系线粒体基因组之间在多个基因区域存在明显差异,为红莲型细胞质雄性不育分子机理研究提供了线索;红莲型不育系与野败型不育系的线粒体基因组之间存在显著差异,马协型不育系与野败型不育系的线粒体基因组之间存在一定差异,在分子水平揭示了不育胞质的多样性。 Abstract:Restriction fragment length polymorphism (RFLP) was used to analyze rice mitochondrial genome.The results indicated obvious differences between mt genome of rice cytoplasmic male sterile line and its maintain line of Honglian type,which further clued CMS mechanism research.Mitochondrial genome difference between sterile line of Honglian type and Wild Abortive type was obvious,some difference between sterile Maxie and Wild Abortive was also detected,it offered molecular evidence for cytoplasmic heterogeneity.     

Characterization and Use of Male Sterility in Hybrid Rice Breeding

The hybrid rice （Oryza sativa L.） breeding that was Initiated In China in the 1970s led to a great improvement in rice productivity. In general, It increases the grain yield by over 20% to the inbred rice varieties, and now hybrid rice has been widely introduced into Africa, Southern Asia and America. These hybrid varieties are generated through either three-line hybrid and two-line hybrid systems; the former is derived from cytoplasmic male sterility （CMS） and the latter derived from genlc male sterility （GMS）. There are three major types of CMS （HL, BT and WA） and two types of GMS （photoperlod-sensitlve （PGMS） and temperature-sensitive （TGMS））. The BT- and HL-type CMS genes are characterized as orf79 and orfH79, which are chimeric toxic genes derived from mltochondrial rearrangement. Rf3 for CMS-WA Is located on chromosome 1, while Rf1, Rf4, Rf5 and Rf6 correspond to CMS-BT, CMSoWA and CMS- HL, located on chromosome 10. The Rfl gene for BT-CMS has been cloned recently, and encodes a mltochondriatargeted PPR protein. PGMS Is thought to be controlled by two recessive loci on chromosomes 7 and 12, whereas nine recessive alleles have been identified for TGMS and mapped on different chromosomes. Attention Is still urgently needed to resolve the molecular complexity of male sterility to assist rice breeding.     

大豆细胞质雄性不育恢复基因GmRf1的精细定位

目前大豆杂交育种主要依赖于以细胞质雄性不育(CMS,cytoplasmic male sterility)为基础的"三系"法,这其中恢复系的选育至关重要.恢复基因的有无和恢复能力的强弱主要通过被测恢复系与不育系测交后代F1的育性确定,既耗时又费力.若能定位和克隆恢复基因(Rf,restorer-of-fertility...     

Expression of mitochondrial gene fragments within the tapetum induce male sterility by limiting the biogenesis of the respiratory machinery in transgenic tobacco

Plant mitochondrial genomes (mtDNAs) are large and undergo frequent recombination events. A common phenotype that emerges as a consequence of altered mtDNA structure is cytoplasmic-male sterility (CMS). The molecular basis for CMS remains unclear, but it seems logical that altered respiration activities would result in reduced pollen production. Analysis of tobacco (Nicotiana tabacum) mtDNAs indicated that CMS-associated loci often contain fragments of known organellar genes. These may assemble with organellar complexes and thereby interfere with normal respiratory functions. Here, we analyzed whether the expression of truncated fragments of mitochondrial genes (i.e. atp4, cox1 and rps3) may induce male sterility by limiting the biogenesis of the respiratory machinery. cDNA fragments corresponding to atp4f, cox1f and rps3f were cloned in-frame to a mitochondrial localization signal and a C-termini HA-tag under a tapetum-specific promoter and introduced to tobacco plants by Agrobacterium-mediated transformation. The constructs were then analyzed for their effect on mitochondrial activity and pollen fertility. Atp4f , Cox1f and Rps3f plants demonstrated male sterility phenotypes, which were tightly correlated with the expression of the recombinant fragments in the floral meristem. Fractionation of native organellar extracts showed that the recombinant ATP4f-HA, COX1f-HA and RPS3f-HA proteins are found in large membrane-associated particles. Analysis of the respiratory activities and protein profiles indicated that organellar complex I was altered in Atp4f, Cox1f and Rps3f plants.     

Molecular markers of nuclear restoration gene Rf1 in sunflower using bulked segregant analysis-RAPD

Restoration of cytoplasmic male sterility (CMS) in sunflower was demonstrated to be controlled by polygenes by analysing 982 effective crosses among 109 self-crossed lines and 16 CMS lines. Two self-crossed lines and one CMS line with distinct genotypes were applied to creation of segregating populations for DNA bulks of the target gene Rfl. Bulked DNA was prepared in order to investigate single gene Rfl and its gene marker among polygenic characters at the same genetic background. Using 80 10-mer operon primers, 620 RAPD reactions were carried out between fertile and sterile DNA bulks. In about 800 loci, primary results showed that 8 were related to the restoration genes. Furthermore. 2 were confirmed as RAPD markers for gene Rfl by examining 9 maintenance and 7 restoration lines. This method is the improvement for bulked segregant analysis[1] with which markers of single gene of target can be identified rapidly among polygenic characters.     

植物细胞核雄性不育的分子基础

植物细胞核雄性育性相关基因克隆,使我们对细胞核雄性育性的了解不仅局限于遗传分析和细胞学水平,而且对细胞核不育的分子基础,以及细胞核不育的分子机理也有了一定的了解。本文旨在对细胞核雄性不育相关研究的进展情况进行简要综述。 Abstract:With the cloning of nuclear male fertility genes,learning of nuclear male fertility not only limited in genetic analysis and cell biology,but also advanced in molecular basis of male nuclear sterility and postulated in possible mechanisms of nuclear male sterility.The objectives of this paper are to review the related progress of plant nuclear male sterility.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

红菜薹雄性不育系花药败育的细胞形态学观察

采用石蜡切片技术,在光学显微镜下系统研究了红菜薹(Brassica campestris L.ssp．chinensis L.var．utilis TsenetLee．)波里马胞质雄性不育系(Polima CMS)、红菜薹萝卜胞质雄性不育系(Ogura CMS)及相应保持系花药发育过程的细胞形态学特征。观察结果表明：红菜薹Polima CMS花药发育受阻于孢原细胞阶段,不形成花粉,属无花粉型,此不育系花药不形成绒毡层和中层;而红菜薹Ogura CMS花药败育发生于小孢子母细胞期或四分体时期,表现为绒毡层细胞异常,挤压四分体,导致四分体和绒毡层同时解体而败育。     

不结球白菜新种质P70-203雄性不育细胞质类型的鉴定

本研究根据OguraCMS、PolimaCMS的不育性状相关的线粒体基因序列设计特异引物,对不结球白菜雄性不育系新种质P70-203及其保持系P60-27-1进行PCR分析.研究结果表明,Polima引物P3/P4,P5/P6在不育系与可育系中均无扩增条带;Ogura引物P1/P2在不育系中扩增出750 bp的特异片段,但可育系中无扩增条带.将扩增的特异条带回收并测序,将得到的测序结果在NCBI中进行Blastn同源性比较,结果与青花菜Ogura(登录号:EU604643)和萝卜Ogura(登录号:AB055438)细胞质雄性不育同源性均达到99%.从分子角度初步推测:该雄性不育系新种质P70-203具有Ogura细胞质.     

RFLP Analysis for Mitochondrial Genome of CMS-Rice

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.     

Synthesis of Ogura male sterile rapeseed (Brassica napus L.) with cold tolerance by protoplast fusion and effects of atrazine resistance on seed yield

Twenty-one cold-tolerant, male sterile Brassica napus somatic hybrids were produced by protoplast fusion. The fusion partners were a coldsensitive, Ogura cytoplasmic male sterile cauliflower inbred (B. oleracea var. botrytis inbred NY7642A) and a cold-tolerant, fertile canola-type B. rapa cv. Candle. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Organellar analyses revealed a very strong bias for Brassica over Raphanus chloroplasts. Cold tolerance was confirmed by cold chamber studies and chloroplast DNA analyses. Good female fertility with 21.4 ± 3.1 seeds/pod was observed in the field using natural pollination vectors. Total seed yield was significantly greater for the atrazine-sensitive somatic hybrids produced in this study than for atrazine-resistant isolines.Abbreviations CMS cytoplasmic male sterility - IA iodoacetate - cpDNA chloroplast DNA     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm

A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.     

大白菜雄性不育系RC7育性相关基因克隆与特性分析

根据orf138的保守序列设计引物,以大白菜萝卜胞质雄性不育系RC7的mtDNA为模板进行PCR扩增,扩增出大小为588 bp的特异条带,该片段在叶片和花蕾中均有表达,没有转录后加工,可编码75个氨基酸,定名为orf75。同源性分析结果表明:orf75推导的氨基酸序列N末端与萝卜Ogu CMS所具有的ORF138一致性为100%,有28个氨基酸完全相同,C末端与钾依赖钠钙交换蛋白-1一致性为54%。初步认为,orf75可能是orf138与钾依赖钠钙交换蛋白-1的编码基因发生重排产生的新的开放阅读框。RC7的不育性与Ogu CMS具有相似性。该588 bp片段还可编码1个含有1个疏水基团和1个跨膜区的67aa的蛋白片段,定名为orf67,属可溶性蛋白。     

细胞质雄性不育水稻线粒体基因组的RFLP分析

利用RFLP技术,比较研究了在农业生产上广泛应用的9种细胞质雄性不育体系的线粒体基因组,结果表明：1）9种水稻雄性不育细胞质的遗传相似性变化范围为0．615～1．000。所有配子体雄性不育细胞质的遗传相似性变化范围为0．6431．000,其中QXA,DY1A和YM15A这3种细胞质的遗传相似性为1．000。所有孢子体雄性不育细胞质的遗传相似性为1．000,2）在配子体细胞质雄性不育水稻中,用3个探针（atpa,atp9＋nad6,cox2）与2种内切酶（HindⅢ,BamHⅠ）的组合未发现不育系与保持系之间的多态性,但在探针atp6,cob,和had2的杂交带型中找到了不育系和保持系之间的一些差异。YTA和YTB在5个探针．内切酶组合（atp61HindⅢ,coblHindⅢ,atp61BamHⅠ,coblBamHⅠ,nad21BamHⅠ）中存在差异。3种细胞质（QX,SJ,DY1）的不育系和保持系之间的差异是相同的,都出现在atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ等3个组合中。YM15A和YM15B在4个组合（atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ,nad2／BamHⅠ）中存在差异。LYA和LYB的差异出现在cob／HindⅢ,cob／BamHⅠ,nad2／BamHⅠ这3个组合中;3）在孢子体细胞质雄性不育水稻中,所有不育系的带型是一样的,所有保持系的带型也一样。不育系和保持系的差异出现在atp6／HindⅢ,cob／HindⅢ,atp6／EcoR,Ⅰcob／EcoRⅠ,cox1／EcoRⅠ,atp6／BamHⅠ,cob／BamHⅠ,cox1／BamHⅠ,cox2／BamHⅠ等9个组合中。这些结果在分子水平上揭示了9种雄性不育细胞质的线粒体基因组存在结构多样性,并为其细胞质雄性不育分子机理的研究打下了基础。