The RING Domain of Rice HEI10 is Essential for Male, But Not Female Fertility.

HEI10 is a conserved E3 ubiquitin ligase involved in crossover formation during meiosis, and is thus essential for both male and female gamete development. Here, we have discovered a novel allele of HEI10 in rice that produces a truncated HEI10 protein missing its N-terminal RING domain, namely sh1 (shorter hei10 1). Unlike previously reported hei10 null alleles that are completely sterile, sh1 exhibits complete male sterility but retains partial female fertility. The causative sh1 mutation is a 76 kb inversion between OsFYVE4 and HEI10, which breaks the integrity of both genes. Allelic tests and complementation assays revealed that the gamete developmental defects of sh1 were caused by disruption of HEI10. Further studies demonstrated that short HEI10 can correctly localise to the nucleus, where it could interact with other proteins that direct meiosis; expressing short HEI10 in hei10 null lines partially restores female fertility. Our data reveal an intriguing mutant allele of HEI10 with differential effects on male and female fertility, providing a new tool to explore similarities and differences between male and female meiosis.

BR signal influences Arabidopsis ovule and seed number through regulating related genes expression by BZR1.

Ovule and seed developments are crucial processes during plant growth, which are affected by different signaling pathways. In this paper, we demonstrate that the brassinosteroid (BR) signal is involved in ovule initiation and development. Ovule and seed numbers are significantly different when comparing BR-related mutants to wild-type controls. Detailed observation indicates that BR regulates the expression level of genes related to ovule development, including HLL, ANT, and AP2, either directly by targeting the promoter sequences or indirectly via regulation by BR-induced transcription factor BZR1. Also, Western blot demonstrates that the dephosphorylation level of BZR1 is consistent with ovule and seed number. The intragenic bzr1-1D suppressors bzs247 and bzs248 have much fewer ovules and seeds than bzr1-1D, which are similar to wild-type, suggesting that the phenotype can be rescued. The molecular and genetic experiments confirm that BZR1 and AP2 probably affect Arabidopsis ovule number determination antagonistically.

[Introduction of Plasmodium falciparum C-terminal region of the merozoite surface protein gene into the chloroplast of tobacco].

OBJECTIVE: To construct chloroplast expression vector, and introduce the C-terminal region of the merozoite surface protein 1 gene (msp1-42) of Plasmodium falciparum 3D7 strain into the chloroplast genome of tobacco for expression of the recombinant protein MSP1-42. METHODS: Forward and reverse primers, adjusted to tobacco chloroplast codon preferences, were used for generation of msp1-42 gene from pBluntmsp plasmid which contains msp1-42 gene. A chloroplast expression vector LRrrmsp was constructed and bombarded into leaves of tobacco by a biolistic He particle delivery system. Media containing 500 mg/L spectinomycin were used for selection of spectinomycin resistant plant. PCR was carried out to check the introduction of the msp1-42 and aadA genes into the chloroplast genome. The transgenic plants with msp1-42 and aadA gene insertion were cut and cultured on the generation MS media containing 500 mg/L spectinomycin for at least 3 turns, and multiple PCR were applied to analyse their homogenization. RESULTS: A chloroplast expression vector LRrrmsp was constructed and confirmed with PCR and enzyme digestion analysis. Six transformants were obtained with a transformation rate 0.6/gun. The msp1-42 and aadA genes were amplified from spectinomycin resistant plants by PCR detection. Wild type chloroplast gene was detected by multiple-PCR analysis. CONCLUSION: A chloroplast expression vector containing msp1-42 gene was constructed. The msp1-42 gene was introduced into chloroplast genome of tobacco and heterogeneous transgenic tobacco was obtained.

Tapetum degeneration retardation is critical for aliphatic metabolism and gene regulation during rice pollen development.

As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR (Tapetum Degeneration Retardation) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC-MS (gas chromatography-mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr. Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total-154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr. In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development.

Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum.

This report provides the complete nucleotide sequences of the full-length cDNA encoding squalene synthase (SQS) and its genomic DNA sequence from a triterpene-producing fungus, Ganoderma lucidum. The cDNA of the squalene synthase (SQS) (GenBank Accession Number: DQ494674) was found to contain an open reading frame (ORF) of 1,404 bp encoding a 468-amino-acid polypeptide, whereas the SQS genomic DNA sequence (GenBank Accession Number: DQ494675) consisted of 1,984 bp and contained four exons and three introns. Only one gene copy was present in the G lucidum genome. The deduced amino acid sequence of Ganoderma lucidum squalene synthase (Gl-SQS) exhibited a high homology with other fungal squalene synthase genes and contained six conserved domains. A phylogenetic analysis revealed that G. lucidum SQS belonged to the fungi SQS group, and was more closely related to the SQS of U. maydis than to those of other fungi. A gene expression analysis showed that the expression level was relatively low in mycelia incubated for 12 days, increased after 14 to 20 days of incubation, and reached a relatively high level in the mushroom primordia. Functional complementation of Gl-SQS in a SQS-deficient strain of Saccharomyces cerevisiae confirmed that the cloned cDNA encoded a squalene synthase.

The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development.

In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.

[Cloning of CTB-PROIN fusion gene and its expression in Escherichia coli].

A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.

Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

Hepatitis B virus core antigen (HBcAg) gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg or core particles) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, Sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. It has been observed that HBcAg was synthesized in yeast cells as a particle consisting of polypeptides with a molecular weight of 21.5 kDa (p21.5). Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (rHBcAg particles) with HBcAg antigenicity mainly located at the densities of 1.27 and 1.40 g ml(-1), respectively. Observation and analysis of the purified rHBcAg products by TEM indicated that rHBcAg peptides could mainly self-assemble into two size classes of core particles. The larger particles were approximately 30.1 nm and the smaller were approximately 21.5 nm in mean diameter. Further observation and analysis of the same rHBcAg (core) particles by AFM also indicated that rHBcAg (core) particles were similar to the native HBcAg (core) particles from infected human hepatocytes and mainly composed of two size classes of partides core. The larger particles were approximately 31.3 nm and the smaller were approximately 22.5 nm in mean diameter which was similar to the results obtained by TEM. All results from both TEM and AFM suggested that core particles (capsids) produced in S. cerevisiae possessed dimorphism.

Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs.

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.

Expression of Choline Oxidase Gene (codA) Enhances Salt Tolerance of the Tobacco.

The codA gene for choline oxidase, which converts choline into betaine. This enzyme, cloned from a soil bacterium Arthrobactor globiformis, has been transferred and expressed in tobacco by the Agrobatcterium-method transformation through the binary plasmid pGAH/codA. The pGAH/codA carried with Km(R) and Hyg(R),and coding sequence for a transit peptide from Rubisco small subunit gene (rbcS) was inserted between 35 S promoter and codA, so that the COD could be introduced into the chloroplast by this transit peptide. The transformed plants were screened on the medium containing the Km and Hyg. PCR, Western and gold immunolocalization tests showed that the codA gene has integrated into the tobacco DNA genome and its protein was expressed and the mature peptide has gone into the chloroplasts by the transit peptide. The results of salt-tolerance measuring for transgenic plants showed that the transgenic plants were more tolerance to the salt than the control plants. The young transgenic plants (1.0--1.5 cm) could survive at 400 mmol/L NaCl MS medium for more than 30 days. From them the higher tolerance plant T4-400 was obtained, which could grow at the 300 mmol/L NaCl MS medium well. The transgenic plants (6--8cm) could grow normally at the 400 mmol/L NaCl MS medium while wild plants failed to do so. So the transferred plant with codA enhanced its tolerance to the salt stress.