[Show abstract][Hide abstract] ABSTRACT: Miniature inverted repeat transposable element (MITE) is one type of transposable element (TE), which is largely found in eukaryotic genomes and involved in a wide variety of biological events. However, only few MITEs were proved to be currently active and their physiological function remains largely unknown.
We found that the amplicon discrepancy of a gene locus LOC_Os01g0420 in different rice cultivar genomes was resulted from the existence of a member of Gaijin-like MITEs (mGing). This result indicated that mGing transposition was occurred at this gene locus. By using a modified transposon display (TD) analysis, the active transpositions of mGing were detected in rice Jiahua No. 1 genome under three conditions: in seedlings germinated from the seeds received a high dose γ-ray irradiation, in plantlets regenerated from anther-derived calli and from scutellum-derived calli, and were confirmed by PCR validation and sequencing. Sequence analysis revealed that single nucleotide polymorphisms (SNPs) or short additional DNA sequences at transposition sites post mGing transposition. It suggested that sequence modification was possibly taken place during mGing transposition. Furthermore, cell re-differentiation experiment showed that active transpositions of both mGing and mPing (another well studied MITE) were identified only in regenerated plantlets.
It is for the first time that mGing active transposition was demonstrated under γ-ray irradiation or in cell re-differentiation process in rice. This newly identified active MITE will provide a foundation for further analysis of the roles of MITEs in biological process.
[Show abstract][Hide abstract] ABSTRACT: To reveal the antagonistic mechanism of B8 strain to Xanthomonas oryzae pv. oryzae, transposon tagging method and chromosome walking were deployed to clone antagonistic related fragments around Tn5 insertion site in the mutant strain B8B. The function of up-stream regulatory sequence of gene 'admA' involved in the antagonistic activity was further identified by gene knocking out technique. An antagonistic related left fragment of Tn5 insertion site, 2 608 bp in length, was obtained by tagging with Kan resistance gene of Tn5. A 2 354 bp right fragment of Tn5 insertion site was amplified with 2 rounds of chromosome walking. The length of the B contig around the Tn5 insertion site was 4 611 bp, containing 7 open reading frames (ORFs). Bioinformatic analysis revealed that these ORFs corresponded to the partial coding regions of glyceraldehyde-3-phosphate dehydrogenase, two LysR family transcriptional regulators, hypothetical protein VSWAT3-20465 of Vibrionales and admA, admB, and partial sequence of admC gene of Pantoea agglomerans biosynthetic gene cluster, respectively. Tn5 was inserted in the up-stream of 200 bp or 894 bp of the sequence corresponding to anrP ORF or admA gene on B8B, respectively. The B-1 and B-2 mutants that lost antagonistic activity were selected by homeologuous recombination technology in association with knocking out plasmid pMB-BG. These results suggested that the transcription and expression of anrP gene might be disrupted as a result of the knocking out of up-stream regulatory sequence by Tn5 in B8B strain, further causing biosythesis regulation of the antagonistic related gene cluster. Thus, the antagonistic related genes in B8 strain is a gene family similar as andrimid biosynthetic gene cluster, and the upstream regulatory region appears to be critical for the antibiotics biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles of appressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST (expressed sequence tag) database of M. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTH11, beta subunit of G protein and SGT1 involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.
Journal of Zhejiang University SCIENCE B 03/2007; 8(2):88-97. · 1.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the changes of G protein-inositol phosphates pathway-related genes and evaluate the role of such changes in the pathogenesis of essential hypertension.
The pressures of the caudal arteries and body weights of 30 spontaneous hypertensive rats (SHRs), 7 two-week-old, 7 4-week-old, 6 six-week-old, 6 eight-week-old, 6 ten-week-old, and 6 twelve-week-old, and 38 normotensive Wistar-Kyoto (WKY) rats, 6 two-week-old, 6 four-week-old, 6 six-week-old, 6 six-week-old, 6 eight-week-old, 7 ten-week-old, and 7 twelve-week-old, were measured. Then the rats were killed and their hearts, aortas, livers, and kidneys were taken out. 294 specimens of total RNA were obtained from the tissues of ventricle of heart, aortic smooth muscle, liver and kidney. RNA array was used to determine the mRNA levels of G proteins G11 and Gq, and phospholipase C-beta (PLCbeta).
The systolic blood pressures of the 6, 8, 10, and 12-week-old SHRs were 158 mm Hg +/- 8 mm Hg, 174 mm Hg +/- 4 mm Hg, 198 mm Hg +/- 13 mm Hg, and 217 mm Hg +/- 9 mm Hg respectively, all significantly higher than those of the age-matched WKY rats (109 mm Hg +/- 6 mm Hg, 128 mm Hg +/- 5 mm Hg,142 mm Hg +/- 4 mm Hg, and 141 mm Hg +/- 5 mm Hg respectively, all P <0.01). The cardiosomatic ratios of the 10- and 12-week-old SHRs were both significantly higher than those of the age-matched WKY rats (both P < 0.01). The G11 mRNA levels in the heart tissue of the 4, 6, 8, 10, and 12-week-old SHRs were 1.42 +/- 0.35, 1.87 +/- 0.40, 1.96 +/- 0.24, 2.09 +/- 0.38, and 2.34 +/- 0.45, all significantly higher than those of the age-matched WKY rats (1.05 +/- 0.18, 1.25 +/- 0.37, 1.26 +/- 0.35, 1.45 +/- 0.30, and 1.51 +/- 0.42 respectively, P < 0.05 or P < 0.01). The Gq mRNA levels of the 4, 6, 8, 10, and 12-week-old SHRs were 1.12 +/- 0.21, 1.30 +/- 0.26, 1.45 +/- 0.35, 1.77 +/- 0.42, and 2.05 +/- 0.46, respectively, all significantly higher than those of the age-matched WKY rats (0.88 +/- 0.09, 0.96 +/- 0.10, 1.03 +/- 0.10, 1.21 +/- 0.38, and 1.29 +/- 0.39 respectively, P < 0.05 or P < 0.01). Similar results were found in the G11 and Gq mRNA levels of the aorta and kidney tissues. The levels of PLCbeta expression in the heart and kidney tissues of the 4, 6, 6, 8, 10, and 12-week-old SHRs were all significantly increased (P <0.05 or P <0.01). Galpha, Gq, and PLCbeta were not significantly expressed in the liver. PLCbeta was hardly found in the aorta.
Increase of the expression of G protein-inositol phosphates pathway-related genes is an important molecular biological mechanism in the pathogenesis and development of essential hypertension.
[Show abstract][Hide abstract] ABSTRACT: To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism.
Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain(s) of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame (ORF) finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrF gene to the mutant B8F strain was used.
A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrF gene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 (the thioesterase domain of type I polyketide synthase) coding region on B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrF gene to the mutant B8F.
The anrF gene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs.
World Journal of Gastroenterology 11/2005; 11(39):6152-8. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AN EXPRESSED SEQUENCE TAGS (EST) resource for tobacco plants (Nicotiana tabacum) was established using high-throughput sequencing of randomly selected clones from 1 cDNA library representing a range of plant organs (leaf, stem, root and root base). Over 5000 ESTs were generated from the 3'-ends of 8000 clones, analyzed by BLAST searches, and categorized functionally. Clustering of these ESTs identified a unigene set of 3600 genes that are represented in the EST collection. 696 unique sequences (19.33% in TUTs) were identified as highly significant matches (E≤10 -20) to the known gene sequences and were regarded as representation of known genes according to results from the BLASTX research of the available database. All annotation ESTs were classified in 18 functional categories, with unique transcripts involved in energy the largest group accounting for 32.32% in all annotation ESTs. After excluding 2450 non-significant TUTs in sequence similarity, 100 unique sequences (1.67% in total TUTs) were identified from N. tabacum database and the remaining 1050 TUTs showed partial homology to genes in other organisms. Through comparing the hybridization data, 359 high-quality ESTs from four tobacco varieties were generated and analyzed using the Cyber-T statistic program, 7 EST from this analysis may have a relation to TMV resistance in tobacco plants, particularly to the Hongda variety. The array results were confirmed using a Real-time quantitative RT-PCR.
[Show abstract][Hide abstract] ABSTRACT: A pair of near isogenic lines G205 and G71 were selected from recombinant inbred lines (RIL) of Zhong156 x Gumei2. On the resistance locus Pi-25(t), G205 had the resistant allele that was from Gumei 2 while G71 had the susceptible allele that was from Zhong156. For the genetic background, different alleles were detected on only 24 loci out of the 672 RFLP or SSLP loci surveyed. The expression profiles of G205 and G71 in response to Magnaporthe grisea were investigated using cDNA microarray containing 2200 Expression Sequence Tags (ESTs). The leaves were inoculated with the pathogen for 12 hours at 4-leaf stage and 998 genes were identified in total. Three genes were up-regulated significantly by the fungus in G205 only. The functions of two genes were known but that of the third gene were unknown. The two genes encoded casein kinase II alpha subunit and retrotransponson TOS17 insertion element respectively. Other thirty-five genes had similar expression patterns between NILs. Among them, 17 genes were up-regulated while 18 genes were down-regulated by the inoculation. The functions of 33 out of the 35 genes were known. BLAST analysis showed that all thirty-five. BLAST analysis showed that all thirty-five genes with known functions were relative to defense reactions, signal transduction, stress response, photosynthesis and sugar metabolism. Northern blot confirmed that four of five differentially displayed genes randomly selected had the same expression patterns as those detected in cDNA microarray. Two of them were up-regulated genes encoding casein kinase II alpha subunit and glycine-rich protein (Grp), and the other two down-regulated genes encoding nitrilase-associated protein and 18S small subnit ribosomal RNA gene respectively. Northern blot also revealed that the expression of Grp was consistently up-regulated from 0 to 36 h after the inoculation of the fungus. These results showed that cDNA microarray was a useful tool to study the molecular mechanisms of disease resistance in plants.