苎麻atp6和atp9基因的克隆表达及与细胞质雄性不育的相关性

摘要: 根据GenBank报道的双子叶植物线粒体atp6和atp9基因编码区保守序列设计简并引物, 通过PCR技术从苎麻细胞质雄性不育系、保持系和恢复系(简称“三系”) mtDNA中扩增目的基因片段, 发现所得序列开放阅读框虽不完整, 但与GenBank报道的其他植物线粒体atp6和atp9基因同源性分别高于94%和85%。采用DNA Walking步移法分别从3′端和5′端扩增两个基因片段的未知侧翼序列, 分离出完整的苎麻线粒体atp6和atp9基因, 包含了完整的开放阅读框。其中“三系”的atp6基因在mtDNA水平、转录和翻译调控水平、蛋白质水平上均无差异。不育系atp9基因在编码区3′端与保持系和恢复系相比存在若干个碱基的差异和缺失; RT-PCR分析还表明, 不育系atp9基因在现蕾期和盛花期的表达量很高。推测不育系atp9基因的结构变异和/或异常表达与苎麻细胞质雄性不育(CMS)的关系密切。     

A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

mtDNA was isolated from cytoplasmic male sterility (CMS) line P3A and its maintainer P3B of kenaf (Hibiscus cannabinus L.). The atp9 gene and its two flanking sequences were obtained using homology cloning and high-efficiency thermal asymmetric interlaced PCR methods. The coding sequences showed only two base pairs difference between the CMS and its maintainer, and shared a homology of over 87 % with atp9 genes from other species in GenBank. However, when comparing the flanking sequences, a 47-bp deletion was characterized at the 3′ flanking sequence of atp9 in the CMS line. Quantitative PCR analysis indicated that the expression level of atp9 in the CMS line was 0.937-fold that of its maintainer. Furthermore, the respiratory rate of anthers in the CMS line was markedly lower than that of its maintainer. The results indicated that the 47-bp deletion at the 3′ flanking sequence of atp9 and/or down-regulated expression of the atp9 gene in the CMS line might be closely related to CMS in kenaf. To confirm whether the 47-bp deletion was specific to cytoplasm of male sterile lines, another 21 varieties were used for further analysis. The results showed that the 47-bp deletion was specific to male sterile cytoplasm (MSC) of kenaf. Based on these, a specific molecular marker was developed to distinguish the MSC from male fertile cytoplasm of kenaf.     

A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice

A fertility restorer gene (Rf-1) of [ms-bo] cytoplasmic male sterility (CMS) in rice has been reported to be responsible for the processing of RNA of aberrant atp6 of mitochondria. We have carried out map-based cloning of the Rf-1 gene and found that a 4.7-kb genomic fragment of a restorer line promoted the processing of aberrant atp6 RNA when introduced into a CMS line. The genomic fragment contained a single open reading frame encoding 18 repeats of the 35 amino acid pentatricopeptide repeat (PPR) motif. The cloned PPR gene is a possible candidate of Rf-1. A non-restoring genotype was identified to have deletions within the coding region.     

Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.)

Molecular markers, coxII SCAR, atp6-2 SCAR and accD-U, have been used for marker-assisted selection of cytoplasmic male sterility (CMS) in pepper. However, the presence of these markers at the sub-stoichiometric level in maintainer lines affects the reliable selection of male sterile (S-) cytoplasm. This study aimed to develop a new CMS-specific molecular marker, SCAR₁₃₀, for reliable identification of S-cytoplasm in pepper, while the new and three previous molecular markers were used to determine the cytoplasm types of pepper lines. Based on mitochondrial genome sequence related amplified polymorphism (SRAP) analysis of the CMS lines and the maintainer lines, SCAR₁₃₀ was developed from a 10-bp deletion at the SRAP primer binding site in the CMS line (130 bp) compared with that in the maintainer line (140 bp). S-cytoplasm could be unambiguously selected from the pepper lines by the different length of the marker bands. Application of the four molecular markers to various pepper lines revealed that SCAR₁₃₀ is more reliable than the other three previous markers, orf507, ψatp6-2 and accD-U. Homology alignment with BLAST showed that the marker was located between trnE and trnS in the Nicotiana tabacum mitochondrial genome. Furthermore, expression of the marker-linked gene was significantly higher at the pollen abortive stage in the CMS line (HW203A) than in the maintainer line, which indicated that the marker was closely related to male sterility. Hence, factors other than orf507 and ψatp6-2 may exist for the regulation of male sterility in pepper.     

紫稻(Oryza sativa L.)线粒体ATP合成酶 atp6 基因转录本 RNA 编辑

紫稻（Oryza sativa L.）细胞质雄性不育系紫稻A是本实验室构建的新型细胞质雄性不育系。本研究使用PCR、RT-PCR、DNA测序等技术,得到了紫稻细胞质雄性不育水稻不育系（樱香A）及其保持系（樱香B）线粒体atp6基因转录本cDNA序列。通过与基因组序列比对发现：樱香Aatp6cDNA序列中,没有发生RNA编辑;而樱香Batp6 cDNA序列中有16个编辑位点,在樱香B cDNA序列16个编辑位点位于15个密码子中,所编码的氨基酸均发生改变：在1003位点由C替换为T,导致原来编码谷氨酰胺密码子（CAA）成为终止密码子（TAA）,保证atp6 mRNA编码一个正常的ATP6多肽;而由于没有发生RNA编辑,樱香A mRNA就不能翻译成正常的多肽。研究表明,RNA编辑在合成正常的ATP6多肽的过程中具有至关重要的作用,同时也说明RNA编辑可能与细胞质雄性不育相关。     

红莲型细胞质雄性不育水稻线粒体atp6基因转录本的编辑位点研究

以红莲（HL）型水稻细胞质雄性不育系A、保持系B及杂种一代F₁为材料,首次比较研究了红莲型水稻线粒体atp6基因转录本的编辑位点及各位点的编辑频率.结果表明atp6基因的转录本有18个编辑位点,其中有15个发生在密码子的第一和第二位点上,这些位点的编辑最终会导致氨基酸种类的变化.18个编辑位点在A、B和F₁中没有差异,但各位点的编辑频率在引入了核恢复基因的条件下发生了较大的变化,完全编辑的比例增加.这些结果首次证明HL型细胞质雄性不育与线粒体atp6转录本的编辑有一定相关性,编辑不充分的转录产物最终会干扰线粒体功能的正常发挥.     

A CMS-Related Gene, Ψatp6-2, Causes Increased ATP Hydrolysis Activity of the Mitochondrial F1Fo-ATP Synthase and Induces Male Sterility in Pepper (Capsicum annuum L.)

Pollen formation is a complex process that is strictly controlled by genetic factors. Although many novel mitochondrial genes have been implicated in the dysfunction of mitochondrial enzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to show that CMS-related genes actually result in the dysfunction of enzyme and little is known about the regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMS lines. Here, we report the characterization of a novel mitochondrial gene, Ψatp6-2, which is hypothesized to play a role in male sterility in pepper. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2 gene in the maintainer line resulted in an increase in ATP hydrolysis activity of the mitochondrial F₁F_o-ATP synthase along with pollen abortion, while silencing the truncated Ψatp6-2 gene in the CMS line resulted in an inhibition of ATP hydrolysis activity and restoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silenced plants to abiotic stresses. Localization experiments showed that premature ATP hydrolysis occurred at the tetrad stage of pollen development in the CMS line, but no ATPase activity was observed in the microspores at the later stage. These results suggest that the Ψatp6-2 gene causes the alteration in ATP hydrolysis activity of the mitochondrial F₁F_o-ATP synthase during pollen development, which eventually leads to male sterility in pepper.     

Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice

Commercial exploitation of heterosis is essential for enhancing productivity of rice. The use of cytoplasmic male sterility (CMS) and fertility restoration system greatly facilitates large scale production of hybrid seed. The wild abortive (WA) cytoplasm is most widely used for hybrid seed production in rice. The present study was undertaken to develop molecular markers for both WA cytoplasm based male sterility and its fertility restoration for use in efficient hybrid breeding. High degree of genetic differentiation of WA-cytoplasm from its normal fertile counterpart was observed due to DNA rearrangements involving five (coxI, coxIII, cob, atp6 and rps3) mitochondrial genes. Cleaved amplified polymorphic sequence (CAPS) markers based on five mitochondrial genes namely, coxIII, cob, atp9, rps3 and 18SrRNA polymorphic between CMS and maintainer line were developed. The utility of these informative markers was demonstrated in purity testing of the CMS line Pusa6A being used in commercial hybrid seed production. Fertility restoration was found to be controlled by a major locus in the Basmati restorer line PRR78, which was mapped to a short marker interval of 0.8 cM and a physical interval of 163.6 kb on rice chromosome 10. A total of 13 pentatricopeptide repeat (PPR) motif containing genes were predicted in a 1.66 Mb region on the long-arm of this chromosome of which, four were present in the marker interval containing the fertility restorer gene. High degree of conservation of gene order was observed between japonica and indica for the predicted PPR genes. A sequence tagged site (STS) and a genic non-coding microsatellite (GNMS) marker were designed based on one of the candidate PPR motif containing genes present in the marker interval, which were validated using F₂ population and other known restorer lines. The candidate gene based marker identified in the present study would be useful in marker assisted selection (MAS) for fertility restorer gene in hybrid breeding programme based on WA-CMS of rice.     

A Comparative Analysis of the <Emphasis Type="Italic">atp8</Emphasis> Gene Between a Cytoplasmic Male Sterile Line and Its Maintainer and Further Development of a Molecular Marker Specific to Male Sterile Cytoplasm in Kenaf (<Emphasis Type="Italic">Hibiscus cannabinus</Emphasis> L.)

In this study, the atp8 gene was cloned from the cytoplasmic male sterile (CMS) line UG93A and its maintainer line UG93B in kenaf. Its DNA sequence analysis showed that atp8 containing 480-bp, encoding 159 amino acid residues, and a 9-bp insertion was found at the 3′flanking sequence in UG93A compared with UG93B. The cDNA sequence of atp8 analyzed by RT-PCR indicated that there were five loci edited, but six loci edited in UG93B. The editing frequencies were higher in sterile cytoplasm than in fertile cytoplasm. The relative expression of atp8 analyzed by real-time PCR showed that the expressed level of atp8 in UG93A was lower than that of its maitainer UG93B and its F₁ hybrid UG93A/992 (a restore line). Furthermore, based on the difference of the 9-bp differences at the 3′flanking sequence of atp8 between UG93A and UG93B, a molecular marker specific to male sterile cytoplasm was developed, which can be used for indentifying whether any germplasm of kenaf is male sterile cytoplasm or male fertile cytoplasm.     

与烟草细胞质雄性不育相关的线粒体基因atp9的mRNA研究

已知导入未编辑atp9 mRNA的烟草表现细胞质雄性不育(CMS),因此认为线粒体基因atp9是引起高等植物CMS的主要基因。为了解atp9在CMS中的作用机制,从3对烟草不育系及其同型保持系中提取atp9,利用实验与理论结合来分析其mRNA在编辑前后以及在不育系及其同型保持系中的一维、三维信息差别。结果表明,atp9mRNA一维信息方面的差异,更重要的是二级结构的差异和稳定性,可能是影响ATP合成而导致CMS的根本原因。     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.