水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育.本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58 000 bp至63 000 bp区域.该基因长4 220 bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性.与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分别为54.9%、54.9%、49.6%和59.5%.经保守结构域搜索发现该蛋白中存在APRT催化结构域.从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明:受温度诱导,水稻"安农S-1"APRT基因的表达变化可能与温敏核雄性不育表现型具相关性.     

水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育。本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58000bp至63000bp区域。该基因K4220bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性。与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分刖为54．9％、54．9％、49．6％和59．5％。经保守结构域搜索发现该蛋白中存在APRT催化结构域。从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明：受温度诱导,水稻“安农S—1”APRT基因的表达变化可能与温敏核雄性不育表现型具相关性。     

Effect of Growth Regulators and Chemicals on Pollen Sterility in TGMS Lines of Rice

Thermosensitive genic male sterility (TGMS) in rice is a widely adopted technique for successful hybrid rice production in Asia. TGMS lines remain male sterile when daily mean temperature is above the critical sterility temperature and are therefore used as female parents. The same line will remain fertile when mean temperature is below the critical sterility temperature. Achievement of 100% male sterility in TGMS lines is important for the successful utilization of TGMS lines as female parents in hybrid rice production. This study examined the external application of some growth regulators and chemicals and their effect on pollen sterility. Among the various treatments, ethrel (800 ppm), salicylic acid (600 ppm) and maleic hydrazide (0.2%) induced a significantly higher percentage of male sterility in the TGMS lines. The sprayed plants also showed higher total phenol accumulation in their flag leaves. The results suggest that it is possible to achieve 100% male sterility in TGMS lines with the external application of growth regulators and chemicals.     

水稻不育系安农S-1育性转换及相关基因的表达分析

通过在自然环境和高温温室内对安农S-1的不同部位进行高温、低温诱导处理,对水稻温敏核不育系安农S-1的温度敏感时期和诱导部位进行了研究。总共进行了8种处理,结果表明：安农S-1的育性转换时期是从花粉母细胞形成到减数分裂的四分体时期之前。在育性转换时期,处于高温的条件下,根部低温处理不能诱导安农S-1可育,穗部低温处理可以使安农S-1保持可育,可见安农S-1的温度敏感部位在幼穗。aprz基因和育性相关,用RT-PCR方法研究了aprt基因在安农S-1不同部位和不同温度环境的表达变化,结果显示,在幼穗中aprt基因的表达在高温环境中被大幅度下调,而在叶中和根中的变化比较小,这说明幼穗对温度最敏感,从侧面验证了引起安农S-1育性转换的温度敏感部位是在幼穗。     

Cloning and Characterization of OsORC2, A New Member of Rice Origin Recognition Complex

In eukaryotic cells, the origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. The isolation, characterization and tissue-specific expression of a putative ORC subunit 2 (OsORC2) in Oryza sativa is described here. A novel cDNA fragment encoding rice ORC2 was isolated by screening the subtractive library, which had a higher expression level in inflorescence meristem than in shoot apical meristem. The full-length cDNA of rice ORC2 was obtained by the method of rapid amplification of cDNA ends, which contained an 1140 bp open reading frame encoding a 379 amino acid polypeptide. Sequence alignment shows that there is a high homology between the deduced amino sequence of OsORC2 and maize ORC2 (85%). The tissue-specific expression pattern of OsORC2 reveals that it is abundant in roots, seedling and inflorescence meristem, while its expression level is much lower in mature leaves and shoot.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsisvia Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and ArabidopsisThese authors contributed equally to this work     

互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

长穗颈温敏核不育水稻穗颈节间长度及细胞学观察

以水稻培矮64S作对照,对隐性长穗颈温敏核不育水稻(Oryza sativa L.)长选3S幼穗各发育时期穗颈节间长度及细胞数量和长度进行比较分析。结果表明,长选3S在幼穗分化的二核期至始花期穗颈节间伸长速度最快,其节间伸长的长度是培矮64S的2.1倍;在花粉母细胞减数分裂期至二核期及始花当天至始花后第3天两个时段,穗颈节间伸长速度慢。穗颈节间的细胞个数和平均长度两者呈现相似的变化规律,但从花粉母细胞减数分裂期至始花期,长选3S穗颈节间薄壁细胞增加3759个,细胞平均长度为72.9μm,培矮64S增加3134个,细胞平均长度为38μm,长选3S穗颈节间的伸长主要是由幼穗分化的花粉母细胞减数分裂期至始花期细胞分裂和细胞伸长共同作用的结果,其中后者的作用更显著。     

温敏核不育系培矮64S及其eui突变体长选3S最上节间蛋白质组差异分析

为从蛋白质表达水平了解长穗颈温敏核不育水稻(Oryza sativa)最上节间伸长机理,采用双向凝胶电泳方法对温敏核不育系培矮64S及其eui突变体长选3S抽穗前2 d的最上节间蛋白质进行了分离,并获得了分辨率和重复性较好的双向凝胶电泳图谱。选取50个差异蛋白质点进行MALDI-TOF-TOF-MS肽质谱指纹图谱分析,从中鉴定出31个已知蛋白质,相对于培矮64S,在长选3S中上调表达的11个蛋白质和下调表达的20个蛋白质。这些差异蛋白质按照其功能可分为7类。这些差异蛋白质可能与长选3S抽穗期最上节间剧烈伸长生长有关,水稻eui基因可能是通过调节抽穗期最上节间这些蛋白质的表达,从而控制最上节间细胞分裂,尤其是细胞的伸长生长。     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi (Brassica campestris L. ssp. chinensis Makino)

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function,whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG)gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

Fine Mapping of Xa2, a Bacterial Blight Resistance Gene in Rice

The rice bacterial blight resistance gene, Xa2, confers resistance to T7147 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. It is located on the long arm of chromosome 4. Here, we report the fine mapping of Xa2 by genetic recombination analysis with simple sequence repeat (SSR) markers according to the genome sequence. Two F₂ populations are constructed to localize Xa2. In a primary analysis with 136 random F₂ plants of Zhenzhuai/IRBB2, it was found that Xa2 was located in approximately 20 cM region. To accurately determine the locus of Xa2, 120 new SSR markers were developed in this region by screening the sequence. Twelve new SSR markers were successfully used in genetic recombination analysis in IR24/IRBB2 population, while 20 in ZZA/IRBB2 population. We found that the nearest SSR markers to Xa2 are HZR950-5 and HZR970-4, which cover approximately 190-kb region. The sequence analysis of this 190-kb region revealed the presence of a homologous sequence of leucine rich repeat (LRR)-kinase. These results are very useful for transferring or pyramiding Xa2 by molecular marker-assistant selection in rice breeding programs and for cloning Xa2 by map-based cloning in combination with a long-range PCR strategy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

甘蓝型油菜分子标记连锁图谱的构建及显性细胞核雄性不育基因的图谱定位

在显性细胞核雄性不育系Rs1046A和双低油菜品种Samourai构建的回交分离群体中,运用AFLP和SSR两种标记技术构建了一个甘蓝型油菜(Brassica napus L.)的分子标记遗传连锁图谱。该图谱共包含138个AFL．P标记、83个SSR标记和1个形态标记,分布于18个主要连锁群、2个三联体和1个连锁对上,图谱总长度为2646cM,偏分离标记的比例为11．7％。显性细胞核雄性不育基因Ms被定位到第10连锁群(LG10)上。同时,偏分离标记聚集于第8连锁群(LG8)和第16连锁群(LGl6)的末端,形成了十分明显的偏分离标记密集区域。研究结果对于油菜核不育两型系的分子标记辅助选择育种具有重要意义,同时也为克隆和分离核不育基因以及研究核不育的分子机理打下了良好的基础。     

Characterization of T-DNA Insertion Mutants and RNAi Silenced Plants of Arabidopsis thaliana UV-damaged DNA Binding Protein 2 (AtUV-DDB2)

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries.     

Inheritance and molecular mapping of two fertility-restoring loci for Honglian gametophytic cytoplasmic male sterility in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

The Honglian cytoplasmic male sterility (cms-HL) system, a novel type of gametophytic CMS in indica rice, is being used for the large-scale commercial production of hybrid rice in China. However, the genetic basis of fertility restoration (Rf) in cms-HL remains unknown. Previous studies have shown that fertility restoration is controlled by a single locus located on chromosome 10, close to the loci Rf1 and Rf4, which respond to cms-BT and cms-WA, respectively. To determine if the Rf locus for cms-HL is different from these Rf loci and to establish fine-scale genetic and physical maps for map-based cloning of the Rf gene, high-resolution mapping of the Rf gene was carried out using RAPD and microsatellite markers in three BCF₁ populations. The results of the genetic linkage analysis indicated that two Rf loci respond to cms-HL, and that these are located in different regions of chromosome 10. One of these loci, Rf5 , co-segregates with the SSR marker RM3150, and is flanked by RM1108 and RM5373, which are 0.9 cM and 1.3 cM away, respectively. Another Rf locus, designated as Rf6(t), co-segregates with RM5373, and is flanked by RM6737 and SBD07 at genetic distances of 0.4 cM. The results also demonstrated these loci are distinct from Rf1 and Rf4. A 105-kb BAC clone covering the Rf6(t) locus was obtained from a rice BAC library. The sequence of a 66-kb segment spanning the Rf6(t) locus was determined by a BLASTX search in the genomic sequence database established for the cultivar 93-11.Communicated by R. Hagemann     

The effect of the recombinant human interleukin-2 Gene in potato (Solanum tuberosum cv. superior)

To examine the effect of the T-cell growth factor (human interleukin-2), we constructed a binary vector, pSSK-1, carrying the recombinant human interleukin-2 (rhlL-2) gene, and transferred it intoAsrobacterium tumefaciens. Using this construct, we then transformed potato explants(Solanum tuberosum cv. Superior), achieving 100% regeneration of shoots on a modified MS medium. Of the putative transformed shoots, 81% rooted and were selected on 200 ms/L kanamycin. Both Southern and northern analyses verified the transformation events. An ELISA test also indicated that the rhlL-2 protein was produced from rhlL-2-transformed potatoes. To determine whether this protein was biologically active in the potato cells, we performed a biological assay using the 11.-2 dependent cell line, CTLL-2. The suspension containing extract from the transformants showed significant proliferation of the 11.-2 dependent CTLL-2 cells, whereas cells did not proliferate in the nontransformed potato. We then grew the verified rhlL-2 transgenic potatoes in soil, and compared their performance with that of nontransgenic potatoes as well as those that had been transformed with GUS. Growth rates, as calculated from plant heights, were up to 50% higher than for either the nontrans-genic or the GUS-transformed potatoes. Similar patterns were found withArabidopsis thaliana plants treated in the same manner. All of these results suggest that rhlLo2 may function as a growth factor in potato.     

Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems

ATP-binding cassette (ABC) proteins constitute a large superfamily found in all kingdoms of living organisms. The recent completion of two draft sequences of the rice (Oryza sativa) genome allowed us to analyze and classify its ABC proteins and to compare to those in Arabidopsis thaliana. We identified a similar number of ABC proteins in rice and Arabidopsis (121 versus 120), despite the rice genome being more than three times the size of Arabidopsis. Both Arabidopsis and rice have representative members in all seven major subfamilies of ABC ATPases (A to G) commonly found in eukaryotes. This comparative analysis allowed the detection of 29 potential orthologous sequences in Arabidopsis and rice. However, plant share with prokaryotes a specific set of ABC systems that is not detected in animals. These ABC systems might be inherited from the cyanobacterial ancestor of chloroplasts. The present work provides the first complete inventory of rice ABC proteins and an updated inventory of those proteins in Arabidopsis.