叶绿体基因组的翻译产物与细胞质雄性不育性

本实验通过对叶绿体基因组翻译产物的分析,发现高粱3197 A不育系及其核代换系白马丁A和遗3 A不育系均比保持系(3197 B)多1个52,000道尔顿的变异多肽。而细胞质来源不同的粒息A不育系除52,000道尔顿多肽外还有1个特有的80,000道尔顿变异多肽。小麦T型不育系具有1个54,000道尔顿的变异多肽。甜菜不育系与保持系无差异。这说明某些作物的细胞质雄性不育性与叶绿体遗传系统有关。在高粱恢复系和F_1叶绿体蛋白质中发现三种特有的多肽。它们是由核基因编码的,这可能是恢复基因的产物。由于恢复基因的作用,在F_1恢复育性的同时其叶绿体变异多肽的合成也受到了强烈的抑制。这进一步证明了不育性与叶绿体的联系。     

高粱线粒体基因组的翻译产物与细胞质雄性不育性

本实验用蛋氨酸标记离体线粒体合成的多肽,用SDS-聚丙烯酰胺凝肢电泳分离并作放射性自显影,对3个不育系及其保持系的分析表明:(1)不育系比保持系多2个特异多肽,分子量为65,000和23,000道尔顿,而且这两个特异多肽在幼苗期与孕穗期表现不同。(2)3197A与白马丁A为两个同质异核不育系,其线粒体合成的多肽完全一致,而与3197A细胞质不同的粒息A不育系则缺少90,000道尔顿以上的高分子量多肽。(3)3197A的母本和F_1也具有不育系中的特异多肽,但合成量显著减弱。由此可以看出,高梁细胞质雄性不育性与线粒体基因组的表达有密切联系。     

普通小麦T型细胞质雄性不育系及其保持系线粒体多肽的电泳比较研究

应用单向SDS-PAGE和双向IEF-SDS电泳技术对两个品种的普通小麦(T.aestivum L.)T型细胞质雄性不育系及其保持系的线粒体多肽进行了比较研究,结论如下:1.黄化苗期不育系和保持系线粒体多肽在单向SDS-PAGE和双向IEF-SDS电泳行为上无明显差别;2.在孕穗期幼穗线粒体多肽的单向SDS-PAGE图谱上,两个不育系都缺少28Kd多肽带纹,因而不育系和保持系间表现出明显的差异。双向IEF-SDS凝胶电泳证实28Kd带纹实际上是分子量相同而等电点分别为5.58和5.65的两个多肽;3.线粒体基因的表达是具有时空性质的;4.线粒体与T型细胞质雄性不育可能存在着某种特定的关系。 本文还就T型细胞质雄性不育的分子机制进行了探索性讨论。     

水稻野败型细胞质雄性不育系和其保持系蛋白质多肽的比较研究

应用单向SDS—PAGE结合蛋白质铬银染色技术对水稻野败型细胞质雄性不育系珍汕97A和其保持系的叶绿体、线粒体和细胞质的蛋白质多肽进行了比较研究,发现两系之间存在明显的差异,生殖器官(穗子)上的差异比营养器官(叶片)上的差异更为显著。在成熟穗上,叶绿体可溶性蛋白不育系有25条带,保持系仅16条带,两者间有19个多肽不同;线粒体可溶性蛋白不育系有28条带,保持系比不育系少30.1和21.8KD两个多肽;细胞质可溶性蛋白丙酮沉淀物的水溶性蛋白组分不育系有24条带,保持系为29条带,两系间却有7条多肽存在差别;细胞质可溶性蛋白丙酮沉淀物的SDS-增溶性蛋白组分不育系有18条带,保持系只有11条带,两者间亦有7条多肽出现差异。由此可以看出,水稻野败型CMS表型的表达可能需要较多个基因的启动和关闭,既与叶绿体和线粒体有关,还涉及到核基因组的作用。     

小伞山羊草(Aegilops umbellata)恢复基因向小麦的转移

鉴定了小伞山羊草(Ae.umbellulata)6条染色体的中国春添加系对T型细胞质雄性不育系育性的影响,发现UAD能较好地恢复T型不育系的育性,表明染色体A上携带有育性恢复基因。添加染色体A在提莫菲维细胞质背景中通过雄配子的传递率为15.6%。同时进一步证明中国春不含有恢复基因。 在体细胞染色体数为42的331个不育系与UAD的杂种衍生后代中选到18个可育株,并对部分植株进行了细胞学鉴定。其中040-5、061-1和061-4与中国春的杂种F_1的育性分离和染色体配对情况表明它们是含有来自小伞山羊草染色体A上的恢复基因的杂合易位系。     

与BT型细胞质雄性不育水稻相关联的双链RNA

以改进的Kemble′s方法提取BT型水稻细胞质雄性不育系及相应保持系线粒体核酸,在电泳分离后的不育系线粒体核酸中发现一特异双链RNA(dsRNA)分子,进一步实验表明,它是线粒体外的成份。与真菌中普遍发现的dsRNA病毒类颗粒(virus-like particles,VLP)相似,这种颗粒也能以细胞质遗传的方式稳定遗传。在表现为杂种优势的F_1代(不育系×恢复系)也发现有这种颗粒,但在F_1代黄化苗相对成熟的基部,此颗粒相对线粒体有减少的趋势,推测是因为F_1核背景不适合此VLP的生存而逐渐被排斥。电镜观察估测dsRNA分子量为6.0×10~6。这种VLP在BT型水稻细胞质雄性不育系中的普遍存在及其遗传行为表明其与不育可能有关。     

高粱A1型细胞质雄性不育系与保持系线粒体基因组分析比较

线粒体基因组易位是导致作物细胞质雄性不育(Cytoplasmic male sterility,CMS)性状产生的重要遗传机制。比较高粱A1型细胞质雄性不育系与保持系线粒体基因组,寻找易位区为克隆高粱A1型细胞质雄性不育相关基因奠定基础。以高粱A1型细胞质雄性不育系Tx623A和其保持系Tx623B为试验材料,采用二代Illumina Hiseq结合三代PacBio测序技术,对2个样品的线粒体基因组进行组装,比较和分析不育系和保持系基因组结构和基因差异。高粱Tx623A和Tx623B线粒体基因组大小分别为449 727 bp和452 772 bp,预测编码开放阅读框(Open reading frame,ORFs)分别为147和145个,且两基因组特有基因分别为8个和6个。两线粒体基因组共线性比较分析,发现存在一个57 kb的基因组片段易位的结构变异(Structural variation,SV)区域,该易位区可能与A1型细胞质雄性不育有关。Tx623A和Tx623B线粒体基因组中易位区为高粱A1型细胞质雄性不育基因克隆提供了基因组信息。     

马协型水稻细胞质雄性不育系线粒体基因组的变异与功能的研究

马协型水稻细胞质雄性不育系是近年培育并广泛应用的一种新型不育系。利用Southern blot、Northern blot和Blue-native PAGE电泳等技术对其线粒体基因组的变异和功能进行研究,分析其雄性不育的分子机理。发现其不育系线粒体基因组中除有一个正常的cox2基因外还存在一个多余的拷贝,且分别转录成不同的转录本。Blue-native PAGE电泳显示,不育系线粒体呼吸链复合物IV的活性明显低于保持系。     

水稻配子体、孢子体细胞质雄性不育系线粒体蛋白质的分析

利用SDS-聚丙烯酰胺凝胶电泳方法分析了水稻配子体细胞质雄性不育系粤泰A、保持系粤泰B、F_1代泰优2号、恢复系胜优2号和孢子体细胞质雄性不育系马协A、保持系马协B、F_1代马协63、恢复系明恢63及另一种孢子体细胞质雄性不育系珍汕97A、保持系珍汕97B、F_1代汕优63、恢复系明恢63黄化苗的线粒体蛋白质。结果表明,粤泰A、B、F_1、恢复系之间出现6条多肽带的差异,马协A、B、F_1、恢复系之间出现4条多肽带的差异,珍汕97A、B、F_1、恢复系之间出现2条多肽带的差异。     

BT型细胞质雄性不育水稻及其三系的线粒体DNA研究

用RAPD技术对BT型水稻胞质雄性不育系秀A及其保持系秀B、恢复系湘晴以及杂种F1代的线粒体DNA进行了比较分析。结果表明不育系与其保持系间存在显著差异;不育系与其F1之间mtDNA也存在差异。在引物OPJ－08的扩增产物中,秀A扩增出一条分子量为800bp的多态性片段,在引物OPK－10的扩增产物中,杂种F1扩增出一条分子量为900bp的片段。把这两片段回收、克隆并制备探针,OPJ－08800的Southern杂交结果显示不育系与其F1杂交图谱存在多态性;OPK－10900的Suthern杂交结果显示不育系与其保持系同存在差异。推测这两片段与育性可能有一定的联系。     

Identification of a Sterility-Inducing Cytoplasm in a Fertile Accession Line of Phaseolus Vulgaris L

Previous investigations into the genetic mechanism of fertility restoration in cytoplasmic male sterile Phaseolus vulgaris suggested that this is a particularly interesting system for the study of nuclear-mitochondrial interactions. This study was conducted to investigate the nature of nuclear-mitochondrial compatibility in fertile accession line G08063, the reported progenitor to the cytoplasmic male sterile line. Results from genetic analysis indicated that fertile line G08063 carried a sterility-inducing cytoplasm with a fertility restoring nuclear genotype. Mitochondrial DNA analysis indicated that the mechanism of fertility restoration by line G08063 was different from that conditioned by Fr, a previously described restorer gene. A mitochondrial DNA sequence associated with sterility and lost upon fertility restoration by nuclear gene Fr was present in the mitochondrial genome of fertile line G08063; this sequence was not carried within the mitochondrial genome of any other P. vulgaris accession line tested.     

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129 ) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST–ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

Comparative proteomics analysis reveals the mechanism of fertility alternation of thermosensitive genic male sterile rice lines under low temperature inducement

Thermosensitive genic male sterile (TGMS) rice line has made great economical contributions in rice production. However, the fertility of TGMS rice line during hybrid seed production is frequently influenced by low temperature, thus leading to its fertility/sterility alteration and hybrid seed production failure. To understand the mechanism of fertility alternation under low temperature inducement, the extracted proteins from young panicles of two TGMS rice lines at the fertility alternation sensitivity stage were analyzed by 2DE. Eighty‐three protein spots were found to be significantly changed in abundance, and identified by MALDI‐TOF‐TOF MS. The identified proteins were involved in 16 metabolic pathways and cellular processes. The young panicles of TGMS rice line Zhu 1S possessed the lower ROS‐scavenging, indole‐3‐acetic acid level, soluble protein, and sugar contents as well as the faster anther wall disintegration than those of TGMS rice line Zhun S. All these major differences might result in that the former is more stable in fertility than the latter. Based on the majority of the 83 identified proteins, together with microstructural, physiological, and biochemical results, a possible fertile alteration mechanism in the young panicles of TGMS rice line under low temperature inducement was proposed. Such a result will help us in breeding TGMS rice lines and production of hybrid seed.     

Development of Anther- defective Cytoplasmic Male Sterile Line from Interspecific Hybridization Between Dongxiang Wild Rice and Cultivated Rice and Preliminary Observations on Its Derivatives

Exploring novel source of cytoplasmic male sterility (CMS) is essential to stablize the productivity of hybrid rice. Dongxiang wild rice ( Oryza rufipogon Griff. ) has been recorded as the northest distributed wild rice in China that is resistant to several biotic or abiotic stresses. A male sterile line M01A defective of anther was identified in the F3 population from an interspecific cross between Dongxiang wild rice and cultivated rice ( Oryza sativa L. ssp. indica ). Crosses and successive backcrosses were made between M01A and a variety of breeding materials and 19 progeny families were obtained. Among the families, some were defective of anthers, and some have twisted and degenerated anthers without microsporogenesis or with a few typical aborted pollen. These resuits implicate that the male sterility of M01A was genetically regulated by the interaction between the nucleus and the cytoplasm. Only one cross produced male-fertile hybrid in which the paternal parent contains a part of the genome of Dongxiang wild rice, which implies that Dongxiang wild rice itself could be the source of the fertility restorer to M01A.     

Fertility restoration in cytoplasmic-nuclear male-sterile lines derived from 3 wild relatives of pigeonpea

Three cytoplasmic-nuclear male-sterile (CMS) lines, one each derived from Cajanus sericeus (A(1) cytoplasm), Cajanus scarabaeoides (A(2) cytoplasm), and Cajanus cajanifolius (A(4) cytoplasm), were crossed to 7 pigeonpea (Cajanus cajan (L.) Millsp.) cultivars in a line x tester mating scheme to study the fertility restoration of the CMS lines. Twenty-one F(1) hybrid combinations were planted in unreplicated 3-row plots in 3 environments. There was no effect of environments on the expression of fertility restoration. Pigeonpea cultivar ICPL 129-3 restored fertility in A(1) cytoplasm and maintained male sterility in the other 2 (A(2) and A(4)) cytoplasms. Among crosses involving CMS line (of A(4) cytoplasm) ICPA 2039 one hybrid combination was male-sterile and another male fertile. The remaining 5 combinations segregated for male-fertility (66-84% fertility restoration). Such testers can easily be purified for use in hybrid breeding programs by selfing and single-plant selection for 2-3 generations.     

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

The so-called ＂wild abortive＂ （WA） type of cytoplasmic male sterility （CMS） derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene（s） remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondriai genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondriai expression profile among the CMS-WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.