Initial Study on Sichuan Apomixis Rice (SAR-1)

SAR-1 is a new germplasm which was discovered in the breeding material of southern multiplication in spring, 1988. The material showing high sterility of pollens is able to set seeds spontaneously. Under isolation, the seed-setting highest rate may reach 55.33%. The florets emasculated by clipping spikelet and lukewarm water still set seeds at certain rates. The completely sterile florets, after being emasculated and checked under microscope one by one, still set seeds, and the highest setting rate is 41.80%. Cytoembryological research indicates that the egg of SAR-1, without fertilization, divides independently into an embryo, which follows the normal process to maturity. Adventitious embryos originate from ovary wall cells. Therefore, it is deduced that SAR-1 has multiple mechanisms of apomixis, and the unfertilized polar nuclei fuse and develop into endosperm cells. The endosperm provids the embryo with nutrient for development. The automatic formation of the endosperm is an obvious feature of SAR-1.     

Identification of WA-Type Three-Line Hybrid Rice with Real-Time Polymerase Chain Reaction (PCR) Method

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R_2-630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R_2-630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P₃, P₄) and TaqMan fluorescence probe (P_3-14) were designed based on the R_2-630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F₁ and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg²⁺ concentration is 3.5 mmol/L.     

INTRODUCING EXOGENOUS DNA INTO PLANTS AFTER POLLINATION——DNA TRAVERSE PATHWAY OF POLLEN TUBE TO REACH EMBRYONIC SAC

The technique of introducing exogenous DNA into plants through the pathway of pollen tube after pollination has been applied since 1978 for transformation of the egg, zygote or carly embryonic cells. By introducing the erogenous DNA containing the goal gene(s) into cotton and rice, several new varieties with economic value and genetic stability are obtained. Thus molecular breeding of crops can be realized by this biotechnique.The pollen tube pathway is demonstrated by tritium-labelled cotton DNA (> 50kb). The labelled DNA is injected into cotton ovaries from the center of the top 24 h after selfing. 30 min to 8 h later, the ovaries are removed. It is clearly shown that the exogenous DNA can only pass from the micrnphyle to the opened nucellus, and then reach the embryonic sac. ~3H-DNA can be found in the embryonic sacs 30 min after injection. In more than 80％ of the embryonic sacs ~3H-DNA can be seen between 2 and 4 h after injection. No ~3H-DNA is found inside the pollen tubes. Therefore it is not th     

EVIDENCE FOR INVOLVEMENT OF PHYTOCHROME REGULATION IN MALE STERILITY OF A MUTANT OF Oryza sativa L.

Abstract— A rice mutant ( Oryza sativa L. Nongken 58S) "Hubei photoperiod-sensitive genie male-sterile rice" ( ms mutant) has been found to be male sterile under long day cycles (LD) and fertile in short day cycles (SD). After formation of the secondary rachis-branch primodia the mutant plants under SD were interrupted in the middle of the long night phase (night break) for 10 days with 5 min pulses of red light (R) or far-red light (FR). Rates of normal pollen and seed setting of the mutant treated by R or R → FR → R declined significantly, while the rates after FR or R → FR treatments were similar to those under SD alone. The result of these induction reversion experiments is consistent with the operational criteria for the involvement of photochrome. Wild-type rice ( O. sativa L. Nongken 58) under the same treatment showed no change in fertility. Experiments on the effect of different dark intervals (20 s to 15 min) between R and FR on male sterility of the ms mutant showed that the longer the dark interval, the greater the escape of R induction from FR reversibility. Treatment with SD or LD after formation of pollen mother cells had no influence on fertility of the ms mutant plants treated previously with R or FR night breaks.     

Participation of the BC Loop in the Correct Folding of Bacteriorhodopsin as Revealed by Solid‐state NMR†

Structural changes in bacteriorhodopsin (bR) in two different processes of retinal reconstitutions were investigated by observing the ¹³C and ¹⁵N solid‐state NMR spectra of [1‐¹³C]Val‐ and [¹⁵N]Pro‐labeled bR. We found that NMR signals of the BC loop were sensitive to changes in protein structure and dynamics, from wild‐type (WT) bR to bacterio‐opsin (bO), regenerated bR and E1001 bR. Regenerated bR was prepared following the addition of retinal into bO obtained from photobleached WT‐bR. E1001 bR was cultured from a retinal‐deficient strain termed E1001 following the addition of retinal to growing cells. ¹⁵N NMR signal at Pro70 in the BC loop in WT‐bR was observed at 122.4 p.p.m., whereas signals were not apparent or partly suppressed in bO and regenerated bR, respectively. Similarly, the ¹³C NMR signal at Val69 in the BC loop at 172.0 p.p.m. that was observed in WT‐bR was significantly decreased in both regenerated bR and bO. These results suggest that the dynamic structure of the BC loop in bO was substantially altered following the removal of retinal. As a consequence, the correct protein structure failed to be recovered via the regenerating process of retinal to bO. On the other hand, ¹³C and ¹⁵N NMR signals at the BC loop in E1001 bR appeared at positions identical to those of WT‐bR. The results of the current study indicate that the BC loop may not always fold correctly in the regenerated bR, which leads to different properties in the regenerated bR compared to that of WT‐bR.     

少精、弱精不育症患者精液中微量元素含量的测定分析

用ICPOES法检测了54例男性少精、弱精不育者精液中Ca、Cd、Cu、Fe、Mg、Mn、Ni、Zn元素的含量,并对相关含量进行数据分析。结果表明,精液中微量元素含量的改变是影响精液质量的重要原因之一。     

Assessment of genetic purity of bulked-seed of rice CMS lines using capillary electrophoresis

Genetically pure cytoplasmic male sterile line (A-line) is essential to generate pure hybrid seeds in order to harness the yield heterosis in rice. Conventionally, seed purity testing is carried by grow-out test, which has many limitations. Seed purity assessments based on molecular markers reduce the time required for analysis significantly. However, it is very tedious as at least 200–400 seeds/seedlings are needed to be analyzed individually. An assay based on bulked-seed and molecular markers will be an ideal system. Keeping these points in view, in the present study, a co-dominant mitochondrial marker was used to test the purity of bulked parental line (A-line) seed utilizing CE system in a genetic analyzer. The results indicate that this method is very simple, accurate, and can be used to test purity of large number of samples rapidly in a cost-effective way compared to grow-out test and conventional molecular marker analysis.     

MPC1001 and its analogues: new antitumor agents from the fungus Cladorrhinum species

[structure: see text] Eight new compounds, MPC1001 and MPC1001B-H, were isolated from the fungus Cladorrhinum sp. KY4922. Multiple NMR experiments and CD data revealed MPC1001 to be an O-methyl derivative of emestrin, a 15-membered antifungal antibiotic containing a unique epidithiodioxopiperazine skeleton. Other compounds were elucidated to be structurally related novel analogues. MPC1001 and the analogues exerted potent antiproliferative activities against a human tumor cell line.     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Nucleation and growth of crazes in amorphous polychlorotrifluoroethylene in liquid nitrogen

The density of mature crazes initially increases linearly with stress and then more rapidly at higher stresses. Once the crazes become observable then density was independent of time. The lowest stress at which an appreciable density of crazes was produced corresponds to the proportional limit. The average velocity of mature crazes was constant for a given stress and varied exponentially with the stress. The velocity depended on stress in the same way that the post-yield point stress depended on strain rate, whereas the yield point varied differently being a nonlinear function of the logarithm of the strain rate. The density of crazes was quantitatively related to the concentration of surface defects at which the crazes nucleate. The craze velocity was directly related to the diffusion coefficient of N₂ into the polymer. The analysis indicates that bulk diffusion of the N₂ governs the craze velocity and that plasticization of the tip of the craze is most important for the nucleation and growth of a craze in PCTFE.     

Roles of gangliosides in mouse embryogenesis and embryonic stem cell differentiation

Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the ma ternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).     

Expression of CTB-human Insulin(BA) Fusion Protein in Gynostemma Pentapyhllum Makino Callus Cells and Its Hypoglycemic Effect in Mice

The plant expression vector of choleratoxin B subunit(CTB)-human insulin(BA) fusion protein pBI121/(CTB-BA) was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express CTB-human insulin fusion protein was constructed and its hypoglycemic effect was evaluated in mice. The plant expression vector pBI121/(CTB-BA) was digested with both BamI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/(CTB-BA) via the freeze tha-wing method, then CTB-BA gene was integrated to G Pentapyhllum Makino callus cells by co-culturing the cells with the transformed LBA4404 strain. The transformed G Pentapyhllum Makino callus cells were identified by DNA se-quence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice.The sequences of synthetic CTB and human insulin genes(BA) were completely identical to those designed. Restriction map proved that the length of gene fragment in-serted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed. The result of RT-PCR was consistent with that expected. The ex-pressed product showed a specific band with a relative molecular mass of 17000 by Western-blot. The human insulin expression amount was 6.03 μIU/mL according to the ELISA result.The animal test showed that only the G Penta-pyhllum Makino callus cell line itself showed activity in decreasing the blood sugar of mice, however, the activity of the transformed G Pentapyhllum Makino callus cells was much higher. The plant expression vector pBI121/(CTB-BA) was constructed and expressed in the G Pentapyhllum Makino callus cells successfully for the first time. The trans-formed G Pentapyhllum Makino callus cells showed high activity in decreasing the blood sugar of mice. This study developed a new way for the development of oral administration insulin.     

Expression Analysis of Rice Pathogenesis-related Proteins Involved in Stress Response and Endophytic Colonization Properties of gfp-tagged Bacillus subtilis CB-R05

Bacillus subtilis CB-R05, possessing antagonistic effects against several fungal pathogens, is a diazotrophic plant growth-promoting bacteria marked with the green fluorescent protein (gfp) gene. To confirm the expression level of the pathogenesis-related (PR) proteins in rice inoculated with CB-R05, the expressions of four pathogenesis-related (PR) proteins (PR2, PR6, PR15, and PR16) were examined in the rice leaves treated with wounding stress over a time period. The PR proteins were generally more strongly expressed in the rice leaves inoculated with CB-R05 compared with the untreated control. The marked gfp-tagged B. subtilis CB-R05 strain was inoculated onto the rice seedlings under axenic conditions. Under the confocal laser scanning microscope (CLSM), the gfp-tagged CB-R05 bacterial cells were observed to penetrate the rhizoplane, especially in the elongation and differentiation zones of the rice roots, and colonize the root intracellularly. The bacteria, 24 h after the gfp-tagged CB-R05 inoculation, were seen to penetrate into the cell wall, cortex, xylem, and concentrate mainly in the vascular bundle. Numerous bacteria were observed within the intercellular spaces, root cortical cells, and xylem vessels. Over time, these bacteria dispersed to the lateral root junctions and propagated slowly from the roots to the stems and leaves. The B. subtilis CB-R05 population in the rice root rhizosphere was also monitored. These results show a very widespread colonization of the B. subtilis CB-R05 in the rice rhizosphere. Further attempts are under way to investigate the competition between the CB-R05 bacteria and the fungal pathogen in vivo.     

Butyltin compounds in vinegar collected in Beijing: Species distribution and source investigation

Forty-eight vinegar samples including white vinegar,rice vinegar and mature vinegar were collected from several markets in Beijing.Butyltin compounds were determined by headspace solid-phase microextraction coupled with gas chromatography and flame photometric detector after in situ ethylation with sodium tetraethylborate.Butyltin species were detected in sixteen vinegar samples and ranged from 0.012 to 14.10 μg Sn L 1.The detection rate of white vinegar is higher than that of rice vinegar and mature vinegar.Vinegar samples with relatively high butyltin concentration(>1.5 μg Sn L 1) were those stored in plastic bags,indicating that the plastic bag was one of the possible sources of butyltin contamination.This was further confirmed by the leaching experiments of three selected plastic bags,and monobutyltin was detected in the leaching solvents.According to the estimation,the average daily intake of total butyltin compounds through vinegar consumption is about 0.04 ng Sn/kg b.w.,much lower than the Tolerable Daily Intake(TDI) of 100 ng Sn/kg b.w.set by the European Food Safety Authority(EFSA).     

CULTURE OF WHEAT PROTOPLAST ——HIGH FREQUENCY MICROCOLONY FORMATION AND PLANT REGENERATION

Embryogenic calli were induced from the mature seeds of the hexaploid semi-winter wheat. The embryogenic cell line was established, and then the friable calli suitable for suspension were induced by adjusting the content of reduced N and changing the 2,4DAA concentration. Protoplasts were isolated from the suspension cells and cultured(?)in KM8P and some other media. A great number of microcolonies were obtained. By altering media, the microcolonies were promoted to grow further and form compact or nodule-like calli. Intact plants were regenerated through organogenesis and embryogenesis on differentiation media.     

Automatic positioning of a microinjector in mouse ES cells and rice protoplasts

A 2-channel (2C) microinjector was prepared by pulling a glass capillary with a theta-shaped cross section. One channel was used as a potential measuring electrode (MeaE) and the other was used as an electrophoretic introduction electrode (IntE). The 2C microinjector was propelled by an oil pressure manipulator driven by a pulse motor, while the MeaE output was recorded continuously. When the 2C microinjector penetrated the cell membrane of a mouse ES cell or a rice protoplast, the output potential changed sharply. The differential of this potential change was used as a stop signal for the pulse motor. Thus, the microinjector was correctly positioned in the cell without losing cell viability. Its success rate was 73% and 84% for ES cells and rice protoplasts, respectively. After the positioning of the microinjector in the cell, Lucifer yellow (LY) was introduced via IntE. Under these conditions, the rate of viable cells was 16% and 62% for ES cells and rice protoplasts, respectively.     

Aptamer-facilitated biomarker discovery (AptaBiD)

Here we introduce a technology for biomarker discovery in which (i) DNA aptamers to biomarkers differentially expressed on the surfaces of cells being in different states are selected; (ii) aptamers are used to isolate biomarkers from the cells; and (iii) the isolated biomarkers are identified by means of mass spectrometry. The technology is termed aptamer-facilitated biomarker discovery (AptaBiD). AptaBiD was used to discover surface biomarkers that distinguish live mature and immature dendritic cells. We selected in vitro two DNA aptamer pools that specifically bind to mature and immature dendritic cells with a difference in strength of approximately 100 times. The aptamer pools were proven to be highly efficient in flow- and magnetic-bead-assisted separation of mature cells from immature cells. The two aptamer pools were then used to isolate biomarkers from the cells. The subsequent mass spectrometry analysis of the isolated proteins revealed unknown biomarkers of immature and mature dendritic cells.     

Novel Pyridyl–Oxazole Carboxamides: Toxicity Assay Determination in Fungi and Zebrafish Embryos

Eight novel pyridyl–oxazole carboxamides were evaluated against fungi and displayed good fungicidal activities against Botrytis cinereal and Rhizoctonia solani. Preliminary bioassay results indicated that at 100 mg/L, compounds 6a–6e, 6g and 6h exhibited 100% fungicidal activities against Botrytis cinerea, and the compound 6b to Rhizoctonia solani at 100%. Then, the zebrafish embryo acute toxicity test was performed to assess the toxicity of 6b and 6c. A series of malformations appeared, when the zebrafish embryos were exposed to 6b and 6c, such as delayed yolk sac resorption, significant shortening of body length, pericardial edema, bending spine, lack of melanin, heart hemorrhage, head hemorrhage, delayed swim sac development, yolk malformation and head malformation. In addition, the acute toxicity of 6b to zebrafish embryo is 4.878 mg/L, and 6c is 6.257 mg/L.     

Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.