The pathogenic site of the C-toxin derived from Bipolaris maydis race C in maize(Zea mays)

Bipolaris maydis race C strain 523 (C523) induces severer leaf blight on cytoplasmic male sterility (CMS)-C maize than on normal (N) maize. Previously, a pathotoxin isolated from C523 (C-toxin) was shown to be responsible for the disease. To understand the basis of the differential responses between CMS-C and N maizes to this fungus, protein synthesisin vitro by mitochondria from N and CMS-C cytoplasms was monitored after their incubation in a solution containing the toxin (0.3%). Similar protein products were detected between the two alloplasmic lines, indicating that the toxin does not directly act on the mitochondrial membrane, nor inhibits the expression of mitochondrial genes. To further locate the action site of the toxin, intact leaves from both N and several subtypes of CMS-C lines were treated by 0.3% toxin. Analysis of electrolyte leakage of leaf cells showed that the leakage rates were similar to one another among the alloplasmic maize lines. In contrast, at a lower concentration of the toxin (0.05%), the leaf cells from CMS-C line were more susceptible to the toxin than those of the other lines. All these results indicate that the target of the toxin action appears to be the cellular-membrane rather than mitochondria, suggesting that the variable susceptibilities toB. maydis between the alloplasmic maize lines might be related to a difference in their cellular-membranes.     

Cytogenetic Mapping of Disease Resistance Genes and Analysis of Their Distribution Features on Chromosomes in Maize

0　IntroductionMaizeisamongthemostintensivelystudiedspeciesingeneticsandoneofagronomicallythemostimportantplants.Therearemanydis easemicrobesandpeststoattackmaize,whichre sultsinlowproductionandbadquality .Withthedevelopmentofverydensegeneticmapconstruc tion ,avarietyoftheimportantdiseaseresistancegenesofmaizeincludingHelminthosporiumtur ciumPassresistancegenesHt1,Htn1andHt2 ,HelminthosporiummaydisNisikresistancegenesRhm1andRhm2 ,maizedwarfmosaicvirusresis tancegeneMdm1,wheatstreakmosaicvi…     

Cloning and identification of a novel human glioma differentiation related geneGDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

Cloning and expression analysis ofMBLL cDNA

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys₃His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys₃His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.     

Cloning of human cytokine signal transduction inhibitor genehumSOCS-2

An EST (gb/AA115239) with high identity to the mouse cytokine signal transduction inhibitor genemmSOCS-2 was selected in GenBank EST database by the homologous screening method. The cDNA with the same sequence of the EST was got in human placenta cDNA library by PCR and a 1011 bp cDNA fragment was selected using above cDNA as probes to perform walking hybridization in placenta cDNA library. The cDNA fragment contains one 594 bp open reading frame (ORF) which encodes 198 amino acid residues. It was proved to be novel after NCBl database screening. Homology comparison showed that this gene has 93% identity tommSOCS-2 at the amino acid level and it has high identities to other related genes in SH₂ domain and SOCS box, so it was namedhumSOCS-2 and the accession number in GenBank is gb/AF020590. The expression analysis showed that the gene is expressed obviously higher in prostate than in other 15 human tissues.     

Molecular cloning and expression analysis of a novel human cDNA fragment encoding a putative Ser/Thr protein kinase

A novel full-length cDNA encoding a putative serine/threonine kinase has been isolated from a human testis cDNA library. A nucleotide sequence of 1225 bp length has been determined containing an open reading frame of 1 044 nucleotides (encoding 348 amino acids). In view of its degree of homology to members of the Ser/Thr protein kinase family and the closest relationship toMus musculus STK-1, the predicted product was designated by the name of HUMSTK-1. Its mRNA is present in large amounts in thymus, and small amounts in testis, small intestine and colon.     

Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA₃ application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity withAAIR genes from other plant species. This cDNA was cloned into expression vector and recombinantE. coli DH5α cells with remarkable AAIR enzyme activity were obtained.     

Cloning of fiber-specific cDNAs and their structural variations in 4 fiber mutants

A mRNA preferentially expressed in cotton fiber was cloned from fiber total RNA of normal upland cotton TM-1 (wild-type) by using RT-PCR and corresponding cDNA (signed as TM-E6) was sequenced. TM-E6 gene had no intron and contained an open reading frame of 771 bp long, and might encode a peptide of 246 amino acids. Other 4 genes, Fl-E6, Li-E6, N-E6 and Bl-E6, which were homologous to TM-E6 gene, were also isolated from 4 fiber mutants of Fiberless Xu-zhou 142, Ligon lintless, Naked seed and Brown lint, respectively. Sequence analysis of each of these mutant genes revealed many variations in structure and nucleotide composition of gene when compared with the sequence of TM-E6 gene. (ⅰ) There was a changeable repetitive segment in which GGCTCA (Gly-Ser) was repeated 3—5 times between the 82nd and the 93rd codons in different mutant genes. Since the change of Gly-Ser repetitive segment occurred not only in the mutants but also in the wild-type cotton, the repeat frequency might not be associated with the mutation of fiber characteristics. (ⅱ) Among the 4 mutant genes, the percentage of changed codons was 7.05% in Fl-E6, 4.98% in Li-E6, and 4.15% in N-E6 and Bl-E6. It seems that the percentage of changed codons in E6 sequence was positively correlated to the degree of fiber morphological variation. (ⅲ) E6 polypeptides of two long-fiberless mutants (Fiberless Xuzhou 142 and Ligon lintless) contained high similar (99.4%) variation in the region of 1—174 amino acids from N-terminus, and those of short-fiberless mutants (Fiberless Xuzhou and naked seed) revealed identical variation in the region of 116th—220th amino acids. It also seems that there was a parallel relation between E6 protein variation and fiber phenotype mutation. (ⅳ) Li-E6 and Bl-E6 genes also expressed at low level in seed coat besides at high level in fiber.     

多重耐药UPEC细菌毒力基因分布特征及进化分型分析

本研究对临床尿路感染病例病原菌耐药情况、毒力基因分布及其进化分型特征,以期为耐药性尿路感染病原防控提供参考。通过对收集的尿样进行细菌分离培养,结合革兰氏染色镜检、16S rDNA对细菌进行鉴定,利用K-B纸片扩散法对细菌进行药敏试验,并通过PCR扩增进行毒力基因及系统发育和进化分型分析。本研究成功检测出20株尿路致病性大肠杆菌（Uropathogenic Escherichia coli, UPEC）,药敏试验显示多数菌株具有多重耐药特征,该批UPEC对青霉素、红霉素、万古霉素耐药率达100%;对羧苄西林、氨苄西林、阿奇霉素耐药率达90%以上;对四环素、诺氟沙星、链霉素、耐药率达80%以上;对环丙沙星、庆大霉素、卡那霉素、氟氧沙星、头孢曲松耐药率达70%以上;对头孢噻肟、呋喃妥因、氯霉素耐药率在35%以上;对阿莫西林与多粘菌素B耐药率低,分别仅为10%与5%。六个毒力基因（iutA、fimH、hly、fyuA、tsh、traT）检出率分别为85%、95%、85%、75%、25%、10%。其系统发育分型中,归属B2群的细菌最多,占比40%(n=8);D群次之,占比35%(n=7);其次为...     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Mapping of two new brown planthopper resistance genes from wild rice

A brown planthopper (BPH) resistance line, B5, derived its resistance genes from the wild riceOryza officinalis Wall exwatt, was hybridized with Taichung Native 1, a cultivar highly susceptible to BPH. A mapping population composed of randomly selected 167 F₂ individuals was used for determining the BPH resistance genes by the restriction fragment length polymorphism analysis (RFLP). Bulked segregant analysis was conducted to identify RFLP makers linked to the BPH resistance genes in B5. The results indicated that the markers linked to BPH resistance are located at two genomic regions on the long arm of chromosome 3 and the short arm of chromosome 4, respectively. The existence of the two loci was further assessed by the quantitative trait locus (QTL) analysis. We located the two loci at a 3.2 cM interval between G1318 and R1925 on chromosome 3 and a 1.2 cM interval between C820 and S11182 on chromosome 4. Comparison with the BPH genes that have been reported indicated that the BPH resistance genes in B5 are novel. These two genes may be useful BPH resistance resource for rice breeding. Furthermore, the mapping of the two genes is useful for cloning the BPH resistance genes.     

Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA end spolymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)^ trail, and included a putative 5‘(98 bp) and a 3‘(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pl of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.     

Cloning and sequencing of cDNA fragment of gene of wheat (Triticum aestivum. L) induced by dehydration

cDNA fragment of the gene (dehydration induced,di1) of wheat (Triticum aestivum. L) induced by 30% PEG-6000 (−1.13 MPa) treatment was isolated with mRNA differential display technique. Northern blot analysis showed that the expression ofdi1 gene improved at 10 h reached the highest at 48 h under 30% PEG-6000 treatment. cDNA fragment ofdi1 gene has been cloned and sequenced (211 bp). DNA sequence analysis shows that there is no homologue in GenBank todi1 cDNA.     

Construction of cDNA library from iron-deficiency induced maize roots and screening and identification of iron-stress geneFdr3

To isolate Fe-deficient related (Fdr) genes, an expression cDNA library of 4.5×10⁵ pfu/μg has been constructed from maize roots in iron-stress. 6 clones have been screened from the cDNA library by differential hybridization screening. It is proved that anFdr3 cDNA clone expressed stronger under iron-deficient condition than under iron-sufficient one by Northern blot and Western blot.     

Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80s ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this riceS4 gene is from a multigene family.     

QTL mapping of resistance to sheath blight in maize（Zea mays L.）

Maize sheath blight (Rhizoctonia Solani) is a widely occurring fungus disease with great harm to corn-pro- ducing regions in the world. The first happening of sheath blight in China was reported in Jilin Province as early as in 1966[1]. Since the 1970s, the enlargement of corn- growing regions, the application of maize hybrids, the increasing use of fertilizers, especially the nitrogenous fertilizer, and a higher growth-density, all have caused a quick spread of sheath blight, the occurring …     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently