Molecular cloning, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice (Orym sativa L.)

By using differential display PCR (DD-PCR) technique, two salt-inducible and one salt-repressed cDNA fragments were isolated from rice. The three cDNA fragments were characterized respectively as partial sequence of rice S-adenosylmethionine decarboxylase (SAMDC) gene, a new member of translation elongation factor 1A gene (namedREF1 A), and a novel gene whose function is unknown (namedSRG1). The full-length cDNA of SAMDC gene (namedSAMDC1) was further isolated by RT-PCR approach and the deduced polypeptide was found to be homologous to SAMDC proteins of other plants, yeast and buman. Northern hybridization revealed that expression of SAMDCl and REFlA was induced, while SRGl was dramatically repressed, by salinity stress. Southern blot analysis demonstrated that SAMDCl and SRGl were present as a single copy gene in rice genome, whereas riceREF1 A gene was organized as a gene family. TheREF1 A,SAMDC1, andSRG1 genes were located on chromosome 3,4, and 6 respectively by RFLP mapping approach using ZYQ8/JX17 DH population and RFLP linkage maps.     

Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA inDunaliella salina (chlorophyta) under hyperosmotic shock

Two subtracted cDNA libraries ofDunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a “driver”, and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as “testers”. The differentially expressed cDNA fragments inD. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments,D27 andD114, were identified from clones pL27 and pL114 after the long-term treatment. Three cDNA fragments,D21, D39, andD88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center inChlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences ofD39, D88, andD114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs inD. salina under salt stress. The expression ofD27, D21, andD88 wasde novo induced by salt stress, and the expression ofD114 andD39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

Linkage mapping of starch branching enzyme III in rice (Oryza sativa L.) and prediction of location of orthologous genes in other grasses

 The chromosomal position of Starch Branching Enzyme III (SBEIII) was determined via linkage to RFLP markers on an existing molecular map of rice (Oryza sativa L.). A cDNA of 890 bp was generated using specific PCR primers designed from available SBEIII sequence data and used as a probe in Southern analysis. The SBEIII cDNA hybridized to multiple restriction fragments, but these fragments mapped to a single locus on rice chromosome 2, flanked by CDO718 and RG157. The detection of a multiple-copy hybridization pattern suggested the possibility of a tandemly duplicated gene at this locus. The map location of orthologous SBE genes in maize, wheat, and oat were predicted based on previously published genetic studies and comparative maps of the grass family. Received : 5 August 1996 / Accepted : 13 September 1996     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.     

Three sucrose transporter genes are expressed in the developing grain of hexaploid wheat

A family of three cDNAs, designated TaSUT1A, 1B and 1D, encoding sucrose transporter (SUT) proteins was isolated from a hexaploid wheat (Triticum aestivum) endosperm library. The cDNA sequences are 96% identical but are distinguishable from one another by virtue of a size polymorphism in the 3-untranslated region (UTR). The predicted amino acid sequences are 98% identical and are highly similar to the sucrose transporters from rice, maize and barley. A gene for TaSUT1 was isolated from genomic libraries of Aegilops tauschii (the donor of the D genome of wheat) and the coding sequence found to be identical to that of TaSUT1D cDNA. There is only one copy of each TaSUT1 gene in hexaploid wheat and it is located on chromosome 4. Genomic Southern analysis and PCR analysis across the 3 polymorphic region of hexaploid, tetraploid and progenitor diploid wheat DNAs established that the TaSUT1A gene was present in the putative A-genome progenitor, T. monococcum, and that the TaSUT1B gene was present in the putative B-genome progenitor, T. searsii. All three TaSUT1 genes are expressed at high levels in filling grain, showing a good correlation with the developmental time course of growth. This reinforces the view that in cereals a major role of SUT1 is in the post-phloem sugar transport pathway associated with seed filling.     

Molecular cloning and differential expression of an γ-aminobutyrate transaminase gene, OsGABA-T, in rice (Oryza sativa) leaves infected with blast fungus

γ-Aminobutyrate transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. Using differential display PCR and cDNA library screening, a full-length GABA-T cDNA (OsGABA-T) was isolated from rice (Oryza sativa) leaves infected with an incompatible race of Magnaporthe grisea. The deduced amino acid sequence comprises 483 amino acid residues and shares 85–69% identity with GABA-T sequences from other plants. OsGABA-T expression is induced by blast fungus infection, mechanical wounding and ultraviolet radiation in rice leaves and is not detected in normal rice organs. This gene is also induced by defense signal molecules such as salicylic acid and abscisic acid, but not by jasmonic acid. Our data suggest that OsGABA-T (GABA shunt) may play a role in restricting the levels of cell death during the host–pathogen interaction.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Heterothallic life cycle in the white root rot fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

To prepare homologous DNA fragments as restriction fragment length polymorphism (RFLP) markers, the genes encoding phenol oxidase, chitinase, and xylanase were amplified from genomic DNA of Rosellinia necatrix strains. RFLP analysis using the amplified DNA fragments as probe was carried out, with segregation of the markers among two sets of F₁ progenies isolated from an independent perithecium. RFLP was frequently found using rpo1 as the RFLP marker among strains of R. necatrix, which was isolated from single ascospores and the circumference of the perithecium. In each set, RFLPs of some F₁ progenies were different from that of the parent strain. Random amplified polymorphic DNA (RAPD) also revealed that several strains, which were of different genotypes from the parent strain, were contained in the single ascospore culture isolated from the same perithecium. From these results, it is suggested that another strain, which was genetically different, was required for mating and development of the ascus in R. necatrix. Therefore, the life cycle in R. necatrix was presumed to be heterothallism. This is the first report about a heterothallic life cycle in R. necatrix.     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Complexity,polymorphism, and connectivity of mouse V k gene families

To define the polymorphism and extent of the mouse immunoglobulin kappa (Igk) gene complex, we have analyzed restriction-enzyme digested genomic DNA from 33 inbred strains of mice with labeled DNA probes corresponding to 16 V _x protein groups (1 of them previously undescribed) and the J _k/C _K region (V, variable; J, joining; C, constant). These probes detected between 1 and 25 distinct restriction enzyme fragments (REF) that appeared in up to eight polymorphic patterns, thus defining eight mouse Jgk haplotypes. The investigated portion of the V _A repertoire was estimated to encompass between 60 and 120 discernable V _k gene-containing REFs. In contrast to mouse V _H gene families, several V _k gene families defined by these probes appeared to overlap. This observation has implications for V _k gene analyses by nucleic acid hybridization and raises the possibility that the V _A gene complex is a continuum of related sequences.Abbreviations used in this paper C constant - Ig immunoglobulin - J joining - REF restriction enzyme fragment - RFLP restriction fragment length polymorphism - V variable     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Transgenic rice plants conferring increased tolerance to rice blast and multiple environmental stresses

We isolated a rice gene (denoted YK1), which showed78 percent amino acid sequence homology to the maize HM1gene. A chimeric gene consisting of a promoter and first intron of maizeubiquitin gene and the cDNA of YK1 was introduced intorice via Agrobacterium mediated transformation. Transgenic riceplants overexpressing this chimeric gene were resistant to rice blast(Magnaporthe grisea) disease, which is one of the mostserious pathogens in rice. Furthermore, the same transgenic plants conferredhigh tolerance to several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide.     

RFLP tagging of a gene for aroma in rice

Summary We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA, and oat cDNA, clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F₃ segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28, on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs.     

Genomic targeting and mapping of tiller inhibition gene (<Emphasis Type="Italic">tin3</Emphasis>) of wheat using ESTs and synteny with rice

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching determines shoot architecture and is a key component of grain yield and/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3A^mL of Triticum monococcum. As a first step towards isolating a candidate gene for tin3, the gene was mapped in relation to physically mapped expressed sequence tags (ESTs) and sequence tag site (STS) markers developed based on synteny with rice. In addition, we investigated the relationship of the wheat region containing tin3 with the corresponding region in rice by comparative genomic analysis. Wheat ESTs that had been previously mapped to deletion bins provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 region. The tin3 gene was mapped to a 324-kb region spanned by two overlapping bacterial artificial chromosomes (BACs) of rice chromosome arm 1L. Wheat–rice synteny was exceptionally high at the tin3 region despite being located in the high-recombination, gene-rich region of wheat. Identification of tightly linked flanking EST and STS markers to the tin3 gene and its localization to highly syntenic rice BACs will assist in the future development of a high-resolution map and map-based cloning of the tin3 gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther’s tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species. Data deposition: The sequence reported in this paper have been deposited in the GeneBank database (Accession No. U12171)     

Expression of a humanS-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.