Isolation of CA/GT Microsatellites from the Paralichthys olivaceus Genome

A library rich in CA/GT microsatellites was constructed from the Paralichthys olivaceus genome by combining biotin capture method and radioactive labeling hybridization. Five hundred and twenty six positive clones were obtained through twice screens. Sequencing confirmed 133 microsatellite loci （number of repeats t〉 5） in 119 positive clones. Of these microsatellites, two （1.5%） had compound repeat motifs, 63 （47.37%） had perfect motifs and 68 （51.13%） had imperfect motifs. Primer pairs were designed in the flanking regions of 22 microsatelites and subjected to PCR amplification. In 8 artificial gynogenesis families, four pairs failed to amplification, one pair was monomorphic, and the rest were polymorphic with an average of 5.2 alleles per locus. Heterozygosities ranged between 0. 375 and 0. 846, PIC ranged between 0. 305 and 0. 823. The results suggested that most of the microsatellites we isolated were qualified to be applied to the population genetic studies of P. olivaceus.

Using Subtracted AFLP to Efficiently Mark an Alien Chromosome Fragment in Wheat Background

为解决AFLP中样品间共同片段对多态性片段识别的干扰 ,作者建立了一种“减法AFLP”标记技术 ,并运用这种技术成功对小麦中的外源染色体片段进行了标记。运用该技术 ,样品间的共同扩增片段显著减少 ,在非变性聚丙稀酰胺凝胶上带型差异十分明显 ,多态性片段很容易分辨。其真实性通过将其中两条成功转换成外源染色体片段特异的SCAR标记得到了验证。减法AFLP标记技术的建立为标记作物中的外源染色体片段提供了一种有效手段。

Solutions to Insecurity Problems in Seed Production of Two-line Hybrid Rice

The insecure problems of seed production have seriously hampered the healthy and sustainable development of two-line hybrid rice.The safety issues on seed production of two-line hybrid rice and current research situation were pointed out in this paper.The three main reasons for unsafety in seed production of twoline hybrid rice were unsuitable site selection,high critical sterility-inducing temperature and the drift of critical temperature.In this paper,strategies and measures were put forward based on many years of practice.It could minimize the risk in seed production of two-line hybrid rice by selecting dual-purpose genic male sterile line with lower critical sterility-inducing temperature and long lower temperature resistant time.Based on the climate data and climatic demands of the ＂three safe-periods＂ in seed production,a new idea for determining appropriate bases and periods for seed production was proposed by using computer technology,which solved the aimless selection of sites and periods for the seed production of two-line hybrid rice.Besides,we established a system of single plant selection and identification method and original seed propagation with cyclic cold water,which could reduce the generation number of original seed propagation in seed production and avoid the drift of critical sterility-inducing temperature.This paper improved the seed production safety in the three aspects of seed nature,seed source and seed production site.

Expression Profiles of TRAIL Receptors and Their Clinical Significance in Human Hepatocellular Carcinoma

Objective To investigate the expression profiles and their clinical significance of TRAIL receptors (TRAILR) in human hepatocellular carcinoma (HCC). Methods The expression profiles of TRAILR were determined in 60 samples from hepatocellular carcinoma, 20 from normal liver tissue and two HCC cell lines HepG2, SMMC-7721 by in situ hybridization. Results Both DR4 and DR5 were present in all HCC tissues as well as normal hepatic tissues. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples (higher DR expression level and lower DcR expression level) were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. The expression level of DR was correlated with HCC differentiation and stage. The weaker expression was more commonly found in HCC with poor differentiation and late stage, while the stronger expression was more common in HCC with middle to high-differentiation and early stage. No relationship was found between DR and gender, age, negative or positive HBsAg, tumor size, grade or metastasis. Multidrug resistance cell lines expressed lower level DR. Conclusion TRAILR expression was prevalent and discrepancy of receptor types was exited in HCC. Loss of DcR1 may contribute for TRAIL therapy for HCC. Key words TRAILR - apoptosis - hepatocellular carcinoma Supported by the Major Fundation of Ministry of Health, NO. 2001–2003

Effect of NGF on c-fos mRNA Expression in Spinal Neuron after Spinal Cord Injury

Objective: To expound the mechanism of nerve growth factor (NGF) to protectspinal cord against injury. Methods: Forty-five rats with 10 g X 2. 5 cm impact T_8 spinal cordinjury (SCI) were divided into 3 groups. The experimental animals received 60 g NGF purified frombovine seminal plasma instantly, 1, 2, 4 h after in jury and an equal volume of normal saline wasgiven to the control group at the same time. The c-fos mRNA levels were detected by in situhybridization. Results: The results showed no evident c-fos expression in normal control group. Thec-fos expression increased markedly in damaged neurons. The peak value of c-fos mRNA arose at 1 h,and c-fos levels in NGF group reduced evidently. Conclusion: NGF could inhibit c-fos expression. Itmay serve as one of the action mechanisms of NGF to protect spinal cord against injury.

An intersection method for locating earthquakes in complex velocity models

The intersection method is one of the basic approaches for locating earthquakes and is not only robust but also efficient. However, its location accuracy is not high, especially for focal depth because the velocity model used for the conventional intersection method is based on homogeneous or laterally homogeneous media, which is too simple. In order to improve the accuracy, we have modified the existing intersection method. In the modified approach, the earthquake loci are not assumed to be circular or hyperbolic and calculation accuracy is improved using a minimum traveltime tree algorithm for tracing rays. The numerical model shows that the modified method can locate earthquakes in complex velocity models.

Great progress in molecular breeding of hybrid rice in CNHRRDC

Elite maintainer lines and restorer lines have been developed by genomic DNA transformation,Analyses of molecular markers, DNA sequences, and Southern blotting have revealed that high DNA polymorphism exists between new developed lines and its receptors, indicating that the special DNA fragment from distant relatives may be integrated into the genome of rice. And several combinations with the potential of super-high yield have been developed from these lines. Therefore, transformation of genomic DNA from distant relatives to the plant of a target receptor may open an avenue for breeding of super-hybrid rice.

Application of Multi-factor Comprehensive Appraisal in Maize Breeding

The study was conducted to evaluate 12 traits of 15 hybrid maize culti- vars using multi-factor comprehensive appraisal. The results showed that Xianyu 335, ND8 and ND2 were better than other cultivars in these agronomic traits. Multi- factor comprehensive appraisal with simple computing, comprehensive and reason- able results is suitable to be used for evaluating the performance of hybrid maize cultivars in field.

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization

AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.

An Innovative Strategy for Reciprocal Distant Hybridization between Spartina alterniflora and Rice

[Objective] This study aimed to explore germplasm resources of salt toler- ant Spartina alterniflora and high yield rice, and develop an innovative strategy for distant hybridization between S. altemiflora and rice. [Method] Crossing （rice ♀ ）： hot water emasculation and glume-cutting pollination method, heat emasculation and pollen-suction pollination method; reciprocal crossing （S. alterniflora ♀ glume-cutting emasculation and pollination method, continuous emasculation-pollination method, hot water emasculation and continuous emasculation-pollination method. [Result] In crossing groups （rice ♀ × S. alterniflora ♂ ）, compared with hot water emasculation and glume-cutting pollination treatment, the number of seed-bearing combinations, seed number per spike and seedling emergence rate in heat emasculation and pollen-suction pollination treatment were improved by 34.09%, 121.21% and 60.07%, respectively; hybridization efficiency was improved by 6-7 times. In reciprocal cross- ing groups （S. altemiflora ♀× rice ♂ ）, compared with glume-cutting emasculation and pollination treatment, the number of seed-bearing combinations and seed num- ber per spike in continuous emasculation-pollination treatment were improved by 3.14 and 4.21 times, respectively; seedling emergence rate was improved by 68.47%; hybridization efficiency was improved by 7-8 times. [Conclusion] Heat emas- culation and pollen-suction pollination method is suitable for hybridization between rice ♀× S. alterniflora ♂; continuous emasculation-pollination method is suitable for hybridization between S. alterniflora ♀× rice ♂.

Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

AIM:To develop a new, rapid and accurate reverse dot blot(RDB) method for the detection of intestinal pathogens in fecal samples.METHODS:The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7, Clostridium botulinum , Bacillus cereus , Clostridium perfringens , Vibrio parahaemolyticus , Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes and Staphylococcus aureus.The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested.Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay.RESULTS:The methods could identify the 12 intestinal pathogens specifi cally, and the detection limit was as low as 103 CFUs.The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%.CONCLUSION:The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.

Agronomic Characters of Elymus dahuricus,Hordeum brevisubulatum and Their BC1F3

[ Objective] This study was to explore the inheritance of BC1 F3 to excellent characteristics of parents（ Elymus dahuricus, Hordeum brevisubu/atum）, heterosis and fertility restoration. E Methodl The agronomic characters such as growth period, growth rate, pollen fertility, fecundity, reproducibility and fresh grass output of Elymus dahudcus, Hordeum brevisubu/atum and BC1 F3 were comparatively studied. E Result l The growth dynamic of the BC1 F3 lined tended to like that of its parent Hordeum brevisubu/atum; although growing periods of various lines were different, they were close to that of Hordeurn brevisubulatum. There were differences in pollen fertility and seed setting, the pollen fertility rate of YF3-93 was higher than recurrent parent Hordeum brevisubulatum, its seed setting rate in open pollination was also higher. Some lines had low pollen fertility rate and seed setting rate, such as the pollen fertility rate of PF3-52. There were significant differences in the output among BC1 F3 lines, e.g. the heterosis （HP） of YF3-64, YF3-74 and YF3-83 were 75.53%, 75.12% and 66.16%, respectively; however, the yields of PF3- 52, PF3-15 and PF3-42 were lower than their parents. ~ Conclusion] This study provided reference for breeding new varieties of forage which is suitable to drought and saline environment.

A REAL-VALUED GENETIC ALGORITHM FOR OPTIMIZATION PROBLEM WITH CONTINUOUS VARIABLES

A real valued genetic algorithm(RVGA) for the optimization problem with continuous variables is proposed. It is composed of a simple and general purpose dynamic scaled fitness and selection operator, crossover operator, mutation operators and adaptive probabilities for these operators. The algorithm is tested by two generally used functions and is used in training a neural network for image recognition. Experimental results show that the algorithm is an efficient global optimization algorithm.

Hybridizing artificial bee colony with biogeography-based optimization for constrained mechanical design problems

A novel hybrid algorithm named ABC-BBO, which integrates artificial bee colony(ABC) algorithm with biogeography-based optimization(BBO) algorithm, is proposed to solve constrained mechanical design problems. ABC-BBO combined the exploration of ABC algorithm with the exploitation of BBO algorithm effectively, and hence it can generate the promising candidate individuals. The proposed hybrid algorithm speeds up the convergence and improves the algorithm's performance. Several benchmark test functions and mechanical design problems are applied to verifying the effects of these improvements and it is demonstrated that the performance of this proposed ABC-BBO is superior to or at least highly competitive with other population-based optimization approaches.

Al_(x)/HKUST-1 prepared by synthetic exchange as catalyst for reduction of NO by CO at low temperature

Al_(x)/HKUST-1(x=1/24,1/12,1/6,1/3),one of the bimetallic copper-based organic framework materials,was successfully prepared by the synthetic exchange method and characterized by X-ray diffraction(XRD),scanning electron microscopy(SEM),specific surface area(BET),thermogravimetric analysis(TG),infrared spectra(IR),X-ray photoelectron spectroscopy(XPS),and H_(2)-temperature programmed reduction(H_(2)-TPR).The findings indicated that Al_(x)/HKUST-1 maintained the octahedral morphology of its precursor(HKUST-1).The thermal stability and catalytic reduction ability of HKUST-1 skeleton were improved by doping aluminum(Al^(3+)).Al_(1/12)/HKUST-1 showed the best performance among all samples,with a nitric oxide(NO)conversion rate of 100%at 210℃(50℃lower than that of HKUST-1).The valence kind of Al,Cu,and O in Al_(1/12)/HKUST-1 did not change after the catalytic reaction,but the contents of Al,Cu,and O in different forms changed significantly.The catalytic process of the Al_(x)/HKUST-1 followed a Langmuir-Hinshelwood mechanism.

Determination of Trace Germanium in Marine Sediments by Hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS)

A method for the analysis of trace germanium in marine sediments by HG-AFS has been investigated. The experimental conditions such as the acidity of reduction reaction, the amount of sodium boro-hydride, the carrier gas flow rate, etc., were tested and optimized by using a kind of orthogonal design. The detection limit of the presented method is 0.95 μg L -1 for germanium. The calibration curve shows a satisfactory line in the concentration range 0-320 μg L -1 Ge with a variation coefficient of ±2.1%.

Simulation of Flux Distribution in Central Metabolism of Saccharo-myces cerevisiae by Hybridized Genetic Algorithm

A scheme of investigating the intracellular metabolic fluxes in central metabolism of Saccharomyces cerevisiae based on isotope model and tracer experiment was developed. The metabolic model applied in this study includes the Embden-Meyerhof-Parnas pathway,the pentose phosphate pathway,the tricarboxylic acid cycle,CO2 anaplerotic reactions,ethanol and acetate formation,and pathways involved in amino acid synthesis. The approach of hybridized genetic algorithm combined with the sequential simplex technique was used to optimize a quadratic error function without the requirement of the information on the partial derivatives. The impact of some key pa-rameters on the algorithm was studied. This approach was proved to be rapid and numerically stable in the analysis of the central metabolism of S.cerevisiae.