Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

Background  

Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.     

Molecular genetic mapping of dwarfing genes in oat

 Restriction fragment length polymorphism (RFLP) analysis provides a valuable tool for characterizing and understanding relationships among genes for useful traits in crop species, particularly in ones with complex genomes such as the hexaploid cultivated oat Avena sativa L. (2n=6x=42). Using Bulked Segregant Analysis (BSA) and F₂ RFLP linkage data, we mapped three dominant oat dwarfing loci to different regions of the oat genome. Dw6, in oat line OT207, is 3.3±1.3 cM from the Xumn145B locus, which has not been placed on the hexaploid oat linkage map. Dw7, in line NC2469-3, is 4.3±2.3 cM from Xcdo1437B and 33±4.1 cM from Xcdo708B. This places Dw7 to linkage group 22. Dw8, in the Japanese lines AV17/3/10 and AV18/2/4, mapped 4.9±2.2 cM from Xcdo1319A in an AV17/3/10בKanota’ F₂ population and 6.6±2.6 cM from it in an AV18/2/4בKanota’ population. This places Dw8 to linkage group 3. Aneuploid analysis of markers linked to the dwarfing genes located Dw6 on the smallest oat chromosome (chromosome 18) and Dw7 on the longest satellited chromosome (chromosome 19). The RFLP markers closely linked to the three dwarfing genes identify distinct regions of the oat genome that contribute to plant height and they should be useful in characterizing new genetic sources of dwarfness in oat. Received: 8 May 1997 / Accepted: 20 May 1997     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

The sequence of a human immunoglobulin epsilon heavy chain constant region gene, and evidence for three non-allelic genes

An immunoglobulin epsilon heavy chain gene was isolated from a DNA library of the human epsilon chain-producing myeloma 266B1 , using a JH gene region probe. The gene was shown to be the one expressed in the myeloma by Southern hybridisation analysis and by comparison of nucleotide sequences with the known amino acid sequence of the epsilon chain made by the myeloma. The gene consists of a variable region segment separated from a constant region segment by a 3.5-kb intervening sequence. The complete sequence of the constant region gene segment shows that this segment is split by intervening sequences into four coding segments corresponding to the four constant region domains of the protein. Using the cloned epsilon constant region gene segment as a probe we obtained evidence, from Southern hybridisation analysis, for three non-allelic epsilon constant region genes. An order on the chromosome for these three genes can be predicted from their pattern of retention in myeloma 266B1 DNA.     

Differential gene expression in salt-tolerant rice mutant and its parental variety

The differential expressions of three genes rbcL, salT and rab!6 in response to ABA, NaCl, PEG and heat shock were investigated in seedlings of a salt-tolerant rice mutant 20 (mutant 20) and its parental variety Oryza sativa var. japonica 77-170(170). By Northern blot analysis it was found that ABA induced the expression of all three genes of rbcL, salT and rab16 in shoots and roots of both 170 and mutant 20 with the exceptions of rab16 in shoots of mutant 20 and rbcL in roots of 170. Lower concentrations of NaCl induced rbcL expression in shoots of mutant 20 but not 170. Higher concentrations of NaCl decreased rbcL expression but induced expressions of salT and rab16 in shoots of both 170 and mutant 20. PEG(15%) and 37℃ heat shock showed almost no effects on the expression of the three genes in mutant 20. However, they caused a decrease in rbcL expression and slight induction of the rab16 gene in 170, with salT expression unaffected. These results indicated that mutant 20 was relatively less responsiv     

Genetic analysis and fine mapping of a novel semidominant dwarfing gene LB4D in rice

A dwarf mutant, designated LB4D, was obtained among the progeny of backcrosses to a wild rice introgression line. Genetic analysis of LB4D indicated that the dwarf phenotype was controlled by a single semidominant dwarfing gene, which was named LB4D. The mutants were categorized as dn-type dwarf mutants according to the pattern of internode reduction. In addition, gibberellin (GA) response tests showed that LB4D plants were neither deficient nor insensitive to GA. This study found that tiller formation by LB4D plants was decreased by 40% compared with the wild type, in contrast to other dominant dwarf mutants that have been identified, indicating that a different dwarfing mechanism might be involved in the LB4D dominant mutant. The reduction of plant height in F(1) plants ranged from 27.9% to 38.1% in different genetic backgrounds, showing that LB4D exerted a stronger dominant dwarfing effect. Using large F(2) and F(3) populations derived from a cross between heterozygous LB4D and the japonica cultivar Nipponbare, the LB4D gene was localized to a 46 kb region between the markers Indel 4 and Indel G on the short arm of chromosome 11, and four predicted genes were identified as candidates in the target region.     

Alteration of nitrogen metabolism in rice variety 'Nipponbare' induced by alkali stress

Aims

Alkali stress (AS) is an important agricultural contaminant and has complex effects on plant metabolism, specifically root physiology. The aim of this study was to test the role of nitrogen metabolism regulation in alkali tolerance of rice variety 'Nipponbare'.

Methods

In this study, the rice seedlings were subjected to salinity stress (SS) or AS. Growth, the contents of inorganic ions, NH ₄ ⁺ -nitrogen (free amino acids), and NO ₃ ^? -nitrogen in the stressed seedlings were then measured. The expression of some critical genes involved in nitrogen metabolism were also assayed to test their roles in the regulation of nitrogen metabolism during adaptation of rice variety 'Nipponbare' to AS.

Results

AS showed a stronger inhibiting effect on rice variety 'Nipponbare' growth than SS. AS may have more complex effects on nitrogen metabolism than SS.

Conclusions

Effects of AS on the nitrogen metabolism of rice variety 'Nipponbare' mainly comprised two mechanisms. Firstly, in roots, AS caused the reduction of NO ₃ ^? content, which caused two harmful consequences, the large downregulation of OsNR1 expression and the subsequent reduction of NH ₄ ⁺ production in roots. On the other hand, under AS (pH, 9.11), almost all the NH ₄ ⁺ was changed to NH₃, which caused a severe deficiency of NH ₄ ⁺ surrounding the roots. Both events might cause a severe deficiency of NH ₄ ⁺ in roots. Under AS, the increased expression of several OsAMT family members in roots might be an adaptative response to the reduction of NH ₄ ⁺ content in roots or the NH ₄ ⁺ deficiency in rhizosphere. Also, the down-regulation of OsNADH-GOGAT and OsGS1;2 in roots might be due to NH ₄ ⁺ deficiency in roots. Secondly, in shoots, AS caused a larger acuumulatiuon of Na⁺, which possibly affected photorespiration and led to a continuous decrease of NH ₄ ⁺ production in shoots, and inhibited the expression of OsFd-GOGAT and OsGS2 in chloroplasts.     

Characteristics of non-allelic interaction of genes gf and B in tomato

Results of investigation on non-allelic interaction of mutant genes gf and B in tomato are represented. It was revealed that gf gene inhibits beta-carotene synthesis owing to incomplete transformation of chlorophyll. In a double BBgfgf homozygote the joint discrete manifestation of effects of two genes takes place resulting in a new phenotype--dirty-orange colour of a fruit.     

A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

Molecular mapping of gibberellin-responsive dwarfing genes in bread wheat

Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes for bread wheat improvement. In this study, the chromosomal locations of several height-reducing genes were determined by screening populations of recombinant inbred lines or doubled haploid lines varying for plant height with microsatellite markers. Linked markers were found for Rht5 (on chromosome 3BS), Rht12 (5AL) and Rht13 (7BS), which accounted for most of the phenotypic variance in height in the respective populations. Large height differences between genotypes (up to 43 cm) indicated linkage to major height-reducing genes. Rht4 was associated with molecular markers on chromosome 2BL, accounting for up to 30% of the variance in height. Confirming previous studies, Rht8 was linked to markers on chromosome 2DS, whereas a population varying for Rht9 revealed a region with a small but significant height effect on chromosome 5AL. The height-reducing effect of these dwarfing genes was repeatable across a range of environments. The molecular markers developed in this study will be useful for marker-assisted selection of alternative height-reducing genes, and to better understand the effects of different Rht genes on wheat growth and agronomic performance.     

水稻优良性状控制基因的定位进展及其在染色体上的分布

利用分子标记可以寻找并且定位目的基因, 分子标记辅助育种技术则能够快速、高效地筛选出携带目的基因的优良品种。文章总结了近年来水稻中已被定位的优良性状控制基因及与其连锁的分子标记, 其中包括抗病虫害、抗寒、抗旱、不育、育性恢复等194个基因, 这些基因中已有14个被克隆测序。在此基础上探讨了这些基因在染色体上的分布趋势。     

Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes

The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR? (hypersensitive reaction—negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category ‘biological process’. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)–scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced in response to cold and gibberellin

Ca²⁺-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca²⁺-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin.