An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.     

New shuttle vectors for ectopic insertion of genes into Bacillus subtilis

We have constructed shuttle vectors for integration of genes via double homologous recombination into three ectopic sites on the chromosome of Bacillus subtilis. The sites of integration are the pyrD, gltA, and sacA genes located at 139 degrees, 172 degrees, and 333 degrees, respectively, on the chromosome. Integration of the vectors into the target genes leads to antibiotic resistance as well as different metabolic phenotypes. B. subtilis strains with integrations of the empty vectors were able to sporulate at rates comparable to wild type cells. Similar levels of expression were obtained from constitutive lacZ fusions integrated at the different sites.     

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with<Emphasis Type="Italic">HMG I/Y</Emphasis> as an endogenous reference gene

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C _T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC _T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Characterization of SLFL1, a pollen-expressed F-box gene located in the Prunus S locus

The S locus and its flanking regions in the genus Prunus (Rosaceae) contain four pollen-expressed F-box genes. These genes contain the S locus F-box genes with low allelic sequence polymorphism genes 1, 2, and 3 (SLFL1, SLFL2, and SLFL3) as well as the putative pollen S gene, named the S haplotype-specific F-box protein gene (SFB). As much less information is available on the function of SLFLs than that of SFB, we analyzed the SLFLs of six S haplotypes of sweet cherry (Prunus avium) in this study. Genomic DNA blot analysis and the isolation of SLFL1 showed that the SLFL1 gene in a functional self-incompatible S ³ haplotype is deleted and only a partial sequence resembling SLFL1 is left in the S ³ locus region, suggesting that SLFL1 by itself is not directly involved in either the GSI reaction or pollen-tube growth. Genomic DNA blot analysis showed that there was no substantial modification or mutation in SLFL2 and SLFL3. A phylogenic analysis of F-box genes in the rosaceous S locus and its border regions showed that Prunus SLFLs were more closely related to maloid S locus F-box brothers than to Prunus SFBs. The functions of SLFLs and the evolution of self-incompatibility in Prunus are discussed based on these results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers, AB360339, AB360340, AB360341, and AB360342, for SLFL1-S ¹ , SLFL1-S ² , SLFL1-S ⁵ , and SLFL1-S ⁶ , respectively.     

The immunoglobulin <Emphasis Type="Italic">IGHD</Emphasis> gene locus in C57BL/6 mice

Four immunoglobulin heavy chain diversity (IGHD) gene subgroups (DFL16, DSP2, DQ52, and DST4) have been identified previously in BALB/c mice. Although the locations of most IGHD genes have been established based on restriction map and Southern blot analysis, a complete mouse IGHD gene locus map at the sequence level is still not available. In addition, a previous restriction fragment length polymorphism study suggested that significant difference in the IGHD gene locus exists between C57BL/6 and BALB/c mice. The author has now analyzed the C57BL/6 mouse genomic data and established a complete map of the IGHD gene locus. All four IGHD subgroups previously identified in BALB/c mice were found to be present in C57BL/6 mice. However, unlike the BALB/c mice, which have at least 13 IGHD genes, the C57BL/6 genome contains only ten IGHD genes, which include one DFL16, six DSP2, one DQ52, and two DST4 genes. There are also differences in the coding regions of the DST4 and DQ52 genes between the two mouse strains.     

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.     

Analysis of two linked genes coding for the acyl carrier protein (ACP) from Arabidopsis thaliana (columbia)

Two linked genes, A1 and A2, coding for nearly identical isoforms of the acyl carrier protein (ACP) were isolated from an Arabidopsis thaliana (columbia) genomic library and sequenced. The amino acids deduced from the nucleotide sequence of the two genes indicate they encode distinct transit peptides, but the mature proteins are the same except for residue 79. Both genes are predicted to contain three introns in similar positions, although they differ in sequence and length. The introns interrupt regions coding for a) the transit peptide, b) the junction of the transit peptide and mature protein, and c) the highly conserved domain surrounding serine 38 to which the phosphopantetheine is attached. Primer extension analysis indicates that at least A1 is active in young plants.     

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.