The indica rice Xieqingzao eB1 (XQZ eB1), a mutant with an elongated uppermost internode, was produced by mutagenesis from elite maintainer line Xieqingzao B (XQZ B) using γ-radiation. Compared with the wild-type XQZ B, plant height and the length of the rice panicle neck, second internode and third internode from the panicle were elongated significantly in XQZ eB1. Genetic analysis indicated that the trait for uppermost internode elongation of XQZ eB1 is controlled by a single recessive gene eui1, which is allelic to that of IR50eui (derived from 76:4512). Further studies were carried out using the bulk segregated analysis approach. An F3 population, derived from the cross XQZ eB1×Aijiaonante, was used to map the mutant gene eui1. Two SSR makers, RM164 and AC9, were identified to link to eui1, their genetic distances to eui1 being 18.4 and 7.9 cM, respectively. The eui1 gene was also mapped to the long arm of chromosome 5 in this research.

Ninety-nine individual plants of a segregating F2 population of a wheat single cross Wen 6×CP90.0.2.4.1 were used in this study to find out whether CP90.0.2.4.1, a derivative of Triticum spelta var. album, carries a novel stripe rust resistance gene other than Yr5. Using molecular marker technology, it was found that only one out of 150 SSR primers could produce amplified polymorphism, and a marker, Xgwm155-147bp, located on chromosome 3A, was found to be linked to the stripe rust resistance gene identified with a genetic distance of 40.5 cM, indicating that the latter was also located on chromosome 3A. Since the only Yr gene previously reported to originate from T. spelta var. album is Yr5, located on 2BL, the only logical inference is that there might be other Yr gene (genes) in T. spelta, and the Yr gene identified in the present study might be a novel one. It was temporarily designated YrSp.

Differences in immunostimulation activity of CpG oligodeoxynucleotides (ODNs) between pigs and mice were studied. In accordance with current research data, three kinds of CpG ODN – 2006, D19 and 1826, which were identified as optimally active in humans, pigs and mice, respectively – and control ODN D48, a non CpG-containing ODN, were selected and synthesized. Results indicated that CpG ODN D19 strongly stimulated the proliferation of porcine peripheral blood mononuclear cells (PBMCs), and the amount of γ-interferon in the supernatant of activated cells increased, exceeding that of all other ODNs tested, while mouse splenocytes did not respond. CpG ODN 1826 strongly stimulated the proliferation of mouse splenocytes, but porcine PBMCs remained unresponsive. CpG ODN 2006 effectively stimulated proliferation of both porcine PBMCs and mouse splenocytes. Therefore some specific CpG ODNs, D19 and 1826, selectively stimulated animal species, while CpG ODN 2006 possessed a common stimulation effect. In addition, CpG ODN D19 predominantly induced the response of type 1 T-helper cells in vivo.

Three Porcine reproductive and respiratory syndrome virus (PRRSV) isolates (HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002) were obtained from pig farms in Hebei and Jiangxi provinces, China. The complete ORF5 gene of the isolates was amplified using RT-PCR and sequenced. It was shown that ORF5 genes of all isolates encoded 200 amino acids. Comparing ORF5 genes of the three isolates and published sequences for five other PRRSV isolates in China, variation analysis showed that all of the isolates were of the American genotype, with 88.2–99.0% amino acid identity. ORF5 genes among BJ-4, S1 and J1 had higher similarity, sharing 98–99% identity of the deduced amino acids. HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002 and CH-1a presented 92–96% identity among their ORF5 genes. Phylogenetic analysis revealed that these isolates could be divided into two subgroups based on the genetic distance of their ORF5 gene: the first subgroup comprised BJ-4, S1 and J1 and was closer to VR2332 and vaccine strains; the second included HB-1(sh)/2002, HB-2(sh)/2002, JX-1/2002 and CH-1a.

The expression pattern of genes encoding putative transcription factors in ethylene signal transduction was investigated, after treatment of tomato (Lycopersicon esculentum cv. UC-82) seedlings with solutions of differing NaCl concentration for 12, 24 and 48 h. The role of ethylene signalling in tomato seedlings adapting to salt stress is discussed. Le-EIL1 gene expression in tomato seedlings was promoted after 12 h of treatment under different NaCl concentrations. The amount of Le-EIL2 transcript increased after 12 and 24 h in 0.08 mol/l NaCl, and the same effect was observed for Le-EIL3 gene expression in tomato seedlings grown in 0.04 and 0.08 mol/l NaCl. After 12 h, Le-ERF1 gene expression was significantly enhanced by 0.04 and 0.08 mol/l NaCl treatments. Le-ERF2 gene transcriptional expression increased with the application of 0.04, 0.08 and 0.16 mol/l NaCl to seedlings. However, Pti4 gene expression did not change remarkably. These results suggest that different temporal gene expression patterns exist in Le-EIL and Le-ERF family members after NaCl stress induction.

The levels of drought tolerance and nucleotide polymorphism at the CBF4 locus were examined in a world-wide sample of 17 core accessions of Arabidopsis thaliana. The results showed that different accessions exhibited considerable differences in adaptation to drought stress. Compared with Columbia accession, the frequency of nucleotide polymorphism at the CBF4 locus of 25av, 203av and 244av accessions, including single nucleotide polymorphism (SNP) and insertion/deletion (Indel), was high, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. No significance in all regions of Tajima's D test indicated that the neutral mutation hypothesis could explain the nucleotide polymorphism in this CBF4 gene region. The higher polymorphism was the result of purification selection. Nucleotide polymorphism in the non-coding region was three times higher than in the coding region. This might indicate a recent relaxation of selection pressures on the non-coding region of CBF4 gene. In the coding region of CBF4, SNP frequency was 1 SNP per 96.4 bp and one non-synonymous mutation was detected from 25av, 203av and 244av accessions: the amino acid variation gly↔val at position 205, caused by the nucleotide variation G↔T at position 1034 (corresponding to the nucleotide at position 19 696 of GenBank accession no. AB015478 as 1). Furthermore, four differential SNPs were discovered in haplotype 6 constituted by 203av, one of them located in the 3′ non-coding region (A↔C at position 1106) and the others in the 5′ non-coding region (A↔G, A↔C and G↔A at positions 27, 129 and 171, respectively). The drought tolerance assay indicated that accession 203av was the best at tolerating water deficiency. We propose that haplotype 6 is consistent with its drought tolerance.

Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, is composed of a single nucleocapsid protein and six genomic ssRNA segments. Based on the known RNA sequence of the RGSV-IR isolate, primers were designed and cDNA of the NS3 gene was obtained by reverse transcription of the virus sense (v) RNA3 and PCR amplification, with genomic RNAs of RGSV-SX isolate as template. The cDNA was then cloned and sequenced. The NS3 gene comprised 588 nt and the sequence identities were 99.1%, 96.2% at the nucleotide level and 98.4%, 96.4% at the amino acid level, compared with those of RGSV-IR and -SC isolates. The 22.9 kDa NS3 protein encoded by RGSV-SX vRNA3 showed 33.0% identity to the 21.6 kDa protein encoded by vRNA5 of RGSV over a span of 80 amino acids, and no other significant matches were found in the GenBank database. The NS3 gene was cloned in pGEX-2T to generate a prokaryotic expression plasmid pGTNS3, and a fusion protein of GST-NS3 at 49.0 kDa was induced in Escherichia coli. Antiserum prepared to this protein reacted with NS3 encoded by RGSV in Western blots. The protein was detected only in infected rice (Oryza sativa), not in purified virus or viruliferous planthopper vectors. Thus NS3 appears to be a protein encoded abundantly only while the virus infects the rice host plant and may be related to the virus pathogenesis.

Embryonic germ (EG) cells are pluripotent cells derived from the primordial germ cells of gonads, gonadal ridges and mesenteries, and analogies of foetuses, with the ability to undergo both self-renewal and multiple differentiation. These cells can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cell of the body. The present paper investigates some factors influencing the efficiency of isolation and culture of human EG cells, such as foetus age, culture serum, added cytokines and feeder cells. The results demonstrate that foetuses of 7–12 weeks are optimal for in vitro culture of human EG cells. The basic medium consisted of DMEM, 1×non-essential amino acids, 2 mM l-glutamine and 1 mM sodium pyruvate. Supplementation with 15% foetal bovine serum, 4 ng/ml human recombinant leukaemia inhibitory factor, 4 ng/ml basic fibroblast growth factor and 20 ng/ml stem cell factor clearly improved the efficiency of isolation and culture of human EG cells. Murine embryonic fibroblasts were better feeder cells than human embryonic fibroblasts, bovine embryonic fibroblasts or STO cell line.

The rabbit defensin gene NP-1 was introduced into embryonic callus cultures of three maize (Zea mays L.) hybrid lines by particle bombardment, and transgenic plants were obtained. Genomic PCR and DNA dot blot analyses confirmed that the NP-1 gene was integrated into the genome of the regenerated T0 maize plants. Genomic PCR and Southern blotting results revealed that the NP-1 gene was transmitted stably from the T0 to the T1 generation. RNA dot blot analysis verified the transcription of the NP-1 gene in T1 plants. When challenged with northern corn leaf blight (Helminthosporium turcicum Pass), the T1 plants expressing the NP-1 gene showed greatly improved resistance to the fungal disease compared with the wild-type maize plants.

A cDNA library was constructed from the heading leaf in the early phase of the heading stage of Chinese cabbage (Brassica rapa L. ssp. pekinensis). By sequencing the randomly selected clones, 1363 sequences longer than 200 bp were found, with better trace data. After removing the poly(A) and contamination sequences, 1162 ESTs longer than 150 bp were obtained, of which 1102 shared significant similarity with known sequences in protein and nucleotide databases of the National Center for Biotechnology Information (NCBI) as revealed by searches using the BLASTX and BLASTN engines. Functional assignment of the ESTs was based on the method used in the Arabidopsis thaliana genome-sequencing project. About 77% of the putative protein sequences with known biological functions best matched with those of A. thaliana deposited in the non-redundant database of NCBI. These data suggest that Chinese cabbage is closely related to A. thaliana. This result is different from that reported in other Brassica species. At nucleotide level, however, 51% of the ESTs were homologous to those deposited for A. thaliana when all ESTs were searched against the est-others database. In addition, 60 ESTs had no homology with any of the plant gene sequences deposited in GenBank. These ESTs are very important for understanding the unique developmental process of Chinesecabbage and elaborating its genetic mapping. Among the genes with assigned functions, the most abundant representatives were those involved in protein synthesis and energy metabolism. With the 1162 ESTs, 895 non-redundant contigs were generated after being aligned using the Seqman II module of DNAStar software at the threshold of more than 80% homology over a minimum of 40 base pairs. Of these, 723 were singletons containing only one EST sequence, indicating that many kinds of such genes are expressed in the heading leaf of Chinese cabbage. An expression profile of Chinese cabbage heading leaf with the 1162 ESTs was therefore acquired in this work. This could be very useful for uncovering the mechanism of the heading process, which is the most obvious characteristic of Chinese cabbage and perhaps other related species, such as Brassica oleracea. This work could accelerate the finding and characterization of genes specifically expressed in the heading stage of Chinese cabbage.

The effects of some factors on the isolation of protoplasts from Penicillium digitatum were studied, including the appropriate material (young mycelia and generating spores), the concentrations of enzyme and osmotic pressure stabilizers, reaction time and reaction temperature. Results demonstrated that germinating spores were an ideal material resource for the isolation of P. digitatum protoplasts. Highest yield and quality of P. digitatum protoplasts were obtained by shaking germinating spores suspended in a solution of 10 mg/ml Lywallzyme™ dissolved in 0.7 M NaCl as osmotic pressure stabilizer at 80 rev/min and 30°C for 3.0–3.5 h. The regeneration rate of the isolated protoplasts was as high as 24.9% on double-layer Czapek medium containing 0.7 M NaCl. Additionally, observation of the protoplast release pattern showed that the protoplasts of P. digitatum were released primarily from the hyphal apex and occasionally from the subapical or original sites of germinating tubes. The protoplasts of P. digitatum were regenerated in a direct manner of either yeast-like cell development or mycelium formation.

Ginseng hairy roots were induced from root explants of ginseng by the co-culture of Agrobacterium rhizogenes and ginseng (Panax ginseng C. A. Mey) roots through direct inoculation. The content of ginsenosides in the ginseng hairy roots after 4 weeks of culture reached the same level as in 3-year-old cultivated ginseng roots, and the content of one ginsenoside (Rb1) increased, showing that left transferred DNA (TL-DNA) has a function of influencing the biosynthesis of ginsenosides. Based on analysis of the RiA4TL-DNA sequence, the rolC gene affecting the synthesis of ginsenosides was amplified and cloned from the ginseng hairy roots by PCR. Comparison with a previously published nucleotide sequence showed that the homology was 99.9%.

The cryptogein (Crypt) gene was obtained by PCR amplification of genomic DNA from Phytophthora cryptogea and confirmed by DNA sequencing. A promoter of the rice phenylalanine ammonia-lyase (PAL) gene was used to regulate the expression of Crypt, because this promoter has a low level of constitutive expression, is strongly induced by pathogen infection and is expressed in epidermal tissues. These promoter characteristics may be suitable for potential inhibition of pathogen attack on epidermal tissues. For functional interaction of Crypt with its outer plasma membrane binding sites, Crypt was led by a signal sequence of the extracellular pathogenesis-related protein (PR1b) of tobacco (Nicotiana tabacum). The fused gene was constructed into a binary vector and the final plasmid (CHF-PAL::Crypt) was transformed into tobacco using the Agrobacterium-mediated transformation method. Twenty-two lines of transformants were obtained from selection medium containing 100 mg/l of kanamycin. Results from PCR amplification and Southern blot analysis demonstrated that Crypt was integrated into the tobacco genome. In the assay of pathogen challenge, nearly two-thirds (68.2%) of the transgenic plants showed significantly enhanced resistance against black shank fungal (Phytophthora parasitica var. nicotianae), brown spot fungal (Alternaria alternata) and wild fire bacterial (Pseudomonas syringae pv. tabaci) diseases. This observation indicates that low-level constitutive expression of Crypt gene in tobacco could have potential use in generating broad-spectrum disease-resistant plants.

Membranous cDNA microarrays containing 2200 unique rice transcripts were designed for screening the characteristics of spatially expressed genes in post-germination rice seedlings. By comparing the profiles obtained, 31 genes were identified as expressed specifically in the plumule, 36 in the mesocotyl and 73 in the radicle. Several genes, such as polyubiquitin, UDP-glucose pyrophosphorylase, sucrose synthase and phosphoglycerate kinase, which encode components of the carbohydrate or protein metabolic reaction cascades, were expressed specifically in the mesocotyl, indicating that degradation reactions of the endospermous reserve starch and proteins occur mainly in the mesocotyl during the post-germination stage. A number of genes involved in defence mechanisms or in the processes of replication, transcription and translation were identified as expressed specifically in the plumule or radicle. Among plumule specifically expressed genes, translation initiation factor 5a, 40s ribosomal protein s28 and ribosomal protein 136 are considered to have a critical role in protein biosynthesis; while allergenic protein, β-D-glucan exohydrolase and actin 11 are genes with defending functions. Among the catalogue of radicle specifically expressed genes, EF-1a, Tat binding protein, replication protein A2, histone h3.2, ribosomal protein s29a and 40s ribosomal protein s19 are genes that function in the process of replication, transcription or translation; whereas glycine-rich protein, wound-induced basic protein, Bowman-Birk proteinase inhibitor and lipid transfer protein-2 are genes involved in defence responses. Results of this experiment have provided insight into post-germination molecular physiology at the genomic level of gene expression.