水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Ｘａ－４紧密连锁的分子标记Ｍ５５的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片ＤＮＡ、半粒种子提取物及Ｘａ－４基因的杂合体ＤＮＡ的ＰＣＲ特异扩增,初步建立了Ｘａ－４的ＰＣＲ标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Ｘａ－４在这些材料中的分布情况。     

The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia

The length polymorphism in the waxy gene, which encodes a granule-bound ADP-glucose-glucosyl transferase [granule-bound starch synthase I (GBSS I), E.C. 2.4.1.11] in barley (Hordeum vulgare), was found. The 5′ leader sequence of the waxy gene of barley germplasm from Japan and Korea was analyzed by the polymerase chain reaction (PCR). The waxy gene of these genetic stocks had three types of length polymorphisms, suggesting that there are insertion/deletion mutations at the 5′ leader sequence of the waxy gene. DNA sequence analysis of the polymorphic PCR products showed that: (1) a 403-bp deletion mutation, which included a complete exon I, was found in the wax allele and a 193-bp insertion sequence was located in the intron I, and (2) the insertion sequence was also located in intron I of the Wax allele. The identity of the insertion sequence was completely conserved between the wax allele and the novel Wax allele. These finding s implying that the wax allele, which was found in indigenous waxy barley, originated in non-waxy barley with the novel Wax allele. Received: 12 January 2001 / Accepted: 17 April 2001     

A practical vector for efficient knockdown of gene expression in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene. These authors contributed equally to this work.     

A new 5′ terminal murine GAPDH exon identified using 5′RACE LaNe

In this work, a ligation-independent, fully gene-specific, nested polymerase chain reaction (PCR) method for the elucidation of 5′ cDNA sequence is described and demonstrated for the first time. Two manifestations of the method, rapid amplification of cDNA ends (RACE) by lariat-dependent nested PCR 5′ (RACE LaNe), at least as simple to perform as conventional RACE, were successfully applied to the murine housekeeping genes phosphoglycerate kinase 1 (PGK1), β-actin (β-ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the alpha thalassemia mental retardation Y homolog (ATRY) gene of the marsupial, Macropus eugenii. Significantly, a new murine GAPDH 5′ exon, separated by 365 kb of intronic sequence from previously annotated GAPDH sequence, was discovered using 5′RACE LaNe.     

Characterization of free and immobilized (<Emphasis Type="Italic">S</Emphasis>)-aminotransferase for acetophenone production

Gordonia amicalis F.5.25.8 has the unique ability to desulfurize dibenzothiophene and to metabolize carbazole [Santos et al., Appl Microbiol Biotechnol 71:355–362, 2006]. Efforts to amplify the dsz genes from G. amicalis F.5.25.8 based on polymerase chain reaction (PCR) primers designed using the dsz gene sequences of Rhodococcus erythropolis IGTS8 were mostly unsuccessful. A comparison of the protein sequences of dissimilar desulfurization enzymes (DszABC, BdsABC, and TdsABC) revealed multiple conserved regions. PCR primers targeting some of the most highly conserved regions of the desulfurization genes allowed us to amplify dsz genes from G. amicalis F.5.25.8. DNA sequence data that include nearly the entirety of the desulfurization operon as well as the promoter region were obtained. The most closely related dsz genes are those of G. alkinovorans strain 1B at 85% identity. The PCR primers reported here should be useful in microbial ecology studies and the amplification of desulfurization genes from previously uncharacterized microbial cultures.     

Inter-simple-sequence-repeat (ISSR) polymorphisms are useful for finding markers associated with disease resistance gene clusters

 We describe a simple and new approach, based on inter-simple sequence repeats (ISSRs), for finding markers linked to clusters of disease resistance genes. In this approach, simple sequence repeats (SSR) are used directly in PCR reactions, and markers found to be linked to disease resistance genes provide important information for the selection of other sequences which can be used with PCR to find other linked markers. Based on an ISSR marker linked to a gene of interest, many new markers can be identified in the same region. We previously demonstrated that ISSR markers are useful in gene tagging and identified a marker, UBC-855₅₀₀, linked to the gene for resistance to fusarium wilt race 4 in chickpea. This ISSR marker provided the information used in the present study for selecting other primers which amplified a region linked to the gene for resistance to fusarium wilt race 4. The primers were based on homology with the (AC)_n sequence and were used for PCR amplifications. Changes in the sequence were at the anchor region of the primers. The repeat (AC)₈T amplified a marker, UBC-825₁₂₀₀, which was located 5.0 cM from the gene for resistance to fusarium wilt race 4 and was closer than other markers. These results indicated that ISSR markers can provide important information for the design of other primers and that by making changes at the 3′ and 5′ anchors close linkage to the desired gene can be found. The approach allows rapid scanning of the targeted region and may provide important information for genome analysis of plant species. Received: 20 January 1998 / Accepted: 19 March 1998     

Comparative analysis in cereals of a key proline catabolism gene.

Proline accumulation and catabolism play significant roles in adaptation to a variety of plant stresses including osmotic stress, drought, temperature, freezing, UV irradiation, heavy metals and pathogen infection. In this study, the gene Δ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH), which catalyzes the second step in the conversion of proline to glutamate, is characterized in a number of cereal species. P5CDH genes from hexaploid wheat, Triticum turgidum (durum wheat), Aegilops tauschii, Triticum monococcum, barley, maize and rice were shown to be conserved in terms of gene structure and sequence, present as a single copy per haploid, non-polyploid genome and located in evolutionarily conserved linkage groups. A wheat cDNA sequence was shown by yeast complementation to encode a functional P5CDH activity. A divergently-transcribed rab7 gene was identified immediately 5′ of P5CDH in all grasses examined, except rice. The rab7/P5CDH intergenic region in these species, which presumably encompasses 5′ regulatory elements of both genes, showed a distinct pattern of sequence evolution with sequences in juxtaposition to each ORF conserved between barley, wheat, A. tauschii and T. monococcum. More distal 5′ sequence in this intergenic region showed a higher rate of divergence, with no homology observed between these regions in the wheat and barley genomes. Maize and rice showed no similarity in regions 5′ of P5CDH when compared with wheat, barley, and each other, apart from a 22 bp region of conserved non-coding sequence (CNS) that is similar to a proline response element identified in the promoter of the Arabidopsis proline dehydrogenase gene. A palindromic motif similar to this cereal CNS was also identified 5′ of the Arabidopsis AtP5CDH gene showing conservation of this sequence in monocot and dicot lineages.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

Genome scanning for resistance-gene analogs in rice, barley, and wheat by high-resolution electrophoresis

 Genes cloned from diverse plants for resistance to different pathogens have sequence similarities in domains presumably involved in pathogen recognition and signal transduction in triggering the defense response. Primers based on the conserved regions of resistance genes often amplify multiple fragments that may not be separable in an agarose gel. We used denaturing polyacrylamide-gel electrophoresis to detect PCR products of plant genomic DNA amplified with primers based on conserved regions of resistance genes. Depending upon the primer pairs used, 30–130 bands were detected in wheat, rice, and barley. As high as 47%, 40%, and 27% of the polymorphic bands were detected in rice, barley, and wheat, respectively, and as high as 12.5% of the polymorphic bands were detected by certain primers in progeny from a cross of the wheat cultivars ‘Stephens’ and ‘Michigan Amber’. Using F₆ recombinant inbred lines from the ‘Stephens’בMichigan Amber’ cross, we demonstrated that polymorphic bands amplified with primers based on leucine-rich repeats, nucleotide-binding sites and protein kinase genes, were inherited as single loci. Linkages between molecular markers and stripe rust resistance genes were detected. This technique provides a new way to develop molecular markers for assessing the genetic diversity of germplasm based upon potential candidate resistance genes in diverse species. Received : 5 September 1997 / Accepted : 6 November 1997     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Rapid detection of Pythium porphyrae in commercial samples of dried Porphyra yezoensis sheets by polymerase chain reaction

The fungal parasite Pythium porphyrae is the causative organism of red rot disease in Porphyra cultivation farms. The detection of P. porphyrae from dried Porphyra yezoensis sheets was achieved using the species-specific primers PP-1 (5′-TGTGTTCTGTGCT-CCTCTCG-3′) and PP-2 (5′-CCCAAATTGGTGTTGCCTCC-3′) with the polymerase chain reaction (PCR). The DNA sequence (707 bp) of PCR product was found to be identical to that amplified from ITS rDNA extracted from a type species of P. porphyrae (IFO 30800, The Institute of Fermentation, Osaka, Japan). Quantities of the product amplified varied with the time when samples were harvested after the occurrence of red rot disease in Porphyra farms. This simple, rapid, and inexpensive method should have great applications in furthering quality control and determination of quality ranking in the Porphyra processing industry.     

Diversity in the Integrase Coding Domain of a gypsy-like Retrotransposon Among Wild Relatives of Rice in the Oryza officinalis Complex

The Oryza officinalis complex is a genetically diverse, tertiary genepool of rice. We analyzed part of the primary structure of the integrase coding domain (ICD) of a gypsy-like retrotransposon from species of the O. officinalis species complex. PCR was performed with degenerate primers that hybridized to conserved sequences in the integrase genes of gypsy-type retrotransposons, using total DNA from different species of the O. officinalis complex as templates. Cloning and sequencing of the PCR products showed that the amplified fragments are highly homologous to each other (75–90%) and belong to one family of retrotransposons that is related to the previously studied RIRE-2 element from rice. Two main subfamilies of 292 and 351 bp were distinguished. Analysis of primary sequence data supports previous reports that sequence divergence during vertical transmission has been the major influence on the evolution of gypsy-type retrotransposons in Oryza species. Based on sequence data phylogenetic relationships among species of the O. officinalis complex were estimated. The data suggests that O. eichingeri is more closely related to the ancestral species of the complex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protential use of PCR-amplified ribosomal intergenic sequences for differentiation of varieties and species ofGossypium cotton

Our purpose was to develop a new approach to the identification ofGossypium cotton varieties and species based on polymerase chain reaction (PCR). Species-specific distinctions within the genusGossypium have been detected by the amplification of ribosomal genes, namely theRrn18-Rrn25 internal transcribed spacer (ITS) regions that had sequence differences. Using the primers to the 3′-end ofRrn18 adjacent to ITS1 and the 5′-end ofRrn25 adjacent to ITS2 from tomato, we have obtained amplified fragments of two cotton species,G. barbadense andG. herbaceum. Interspecies distinctions have been revealed by the restriction assay of these amplification products. The restriction patterns are distinguished not only by number but by location and intensity of the bands. Our results illustrate the effective use of differences in ribosomal intergenic sequences for the differentiation of varieties and species ofGossypium.     

Global analysis of gene expression by differential display

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. However, there has been lack of an accurate guidance on how many DD polymerase chain reaction (PCR) primer combinations are needed to display most of the genes expressed in a eukaryotic cell. This study critically evaluated the gene coverage by DD as a function of the number of arbitrary primers, the number of 3′ bases of an arbitrary primer required to completely match an mRNA target sequence, the additional 5′ base match(s) of arbitrary primers in first-strand cDNA recognition, and the length of mRNA tails being analyzed. The resulting new DD mathematical model predicts that 80 to 160 arbitrary 13mers, when used in combinations with 3 one-base anchored oligo-dT primers, would allow any given mRNA within a eukaryotic cell to be detected with a 74% to 93% probability, respectively. The prediction was supported by both computer simulation of the DD process and experimental data from a comprehensive fluorescent DD screening for target genes of tumor-suppressor p53. Thus, this work provides a theoretical foundation upon which global analysis of gene expression by DD can be pursued.     

Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation

Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in the 3′ untranslated region (3′ UTR), in wheat plants grown at 20°C and cold-acclimated for 1–4 weeks at 2°C. The amino acid variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3′ UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression, suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat.     

Development of microsatellite markers from tartary buckwheat

A genomic library enriched with (gT)_n repeats from tartary buckwheat (Fagopyrum tataricum) was constructed using 5′-anchored PCR for the development of microsattellite markers. Sequencing analysis of 5 clones from the library showed that they all contained microsatellites (totally 10 loci), and each was unique. An additional locus-specific primer was designed according to flanking sequence. Two of the microsatellite loci of 10 tartary buckwheat varieties were amplified using an anchored primer and a locus-specific primer, which revealed a clear polymorphic pattern. The data confirmed that the degenerate primer was reliably anchoring at the 5′-end of the microsatellite, and the primers developed based on this technology could be used for diversity analysis of tartary buckwheat.     

Fine Mapping and in silico Isolation of the EUI1 Gene Controlling Upper Internode Elongation in Rice

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9804 bp long, and comprises two exons and one intron. The length of the cDNA is 1931 bp containing a 1734 bp ORF, a 110 bp 5′-UTR and a 87 bp 3′-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. Hongli Ma, Shubiao Zhang: These authors contributed equally to this work     

Target region amplification polymorphism: A novel marker technique for plant genotyping

The advent of large-scale DNA sequencing technology has generated a tremendous amount of sequence information for many important organisms. We have developed a rapid and efficient PCR-based technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. This target region amplification polymorphism (TRAP) technique uses 2 primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database; the second primer, the arbitrary primer, is an arbitrary sequence with either an AT-or GC-rich core to anneal with an intron or exon, respectively. PCR amplification is run for the first 5 cycles with an annealing temperature of 35°C, followed by 35 cycles with an annealing temperature of 50°C. For different plant species, each PCR reaction can generate as many as 50 scorable fragments with sizes ranging from 50–900 bp when separated on a 6.5% polyacrylamide sequencing gel. The TRAP technique should be useful in genotyping germplasm collections and in tagging genes governing desirable agronomic traits of crop plants.