Isolation and Structural Analysis of the Seed-Specific Promoter from Soybean

The promoter region (BCSP666) of β-conglycinin α-subunit gene from the genomic DNA of soybean Jilin 43 was isolated by PCR method. Sequencing analysis showed that the cloned fragment BCSP666 had the similar structure to the soybean seed-specific promoter β-conglycinin α'-subunit gene promoter and β-conglycinin β-subunit gene promoter, and it also contains many motifs that contribute to the seed-specific promoter activity. Based on this sequencing analysis, we deduced that promoter fragment BCSP666 had the seed-sepecific promoter activity. And then we constructed the seedspecific expression vector pBMI666 with the promoter fragment BCSP666 and △6-fatty acid desaturase gene from Mortierella isabellina. The △6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in the production of a human essential fatty acid, γ-linolenic acid(GLA). The production of γ-linolenic acid(GLA) was observed in soybean callus cells, which were transformed with this vector. This confirmed the activity of the activity fragment BCSP666.     

Enhancement of Arachidonic Acid Production by Mortierella isabellina Through Protoplast Regeneration Mutagenesis

A developed method was used for the enhancement of arachidonic acid production by M. isabellina. An orthogonal, rotatable and central composite design was applied to determine the optimum conditions for protoplast regeneration mutagenesis. The results showed that a commixture enzyme (cellulase and glusulase) at the concentration of 4%, enzymolysis temperature at 30℃ and enzymolysis time on 7.5 h were the optimal conditions, in which the lethality of M. isabellina spores was 78.4%. After mutagenesis and re-s...     

Gene Expression Profiling Related to Hyphal Growth in a Temperature-Sensitive Mutant of Magnaporthe oryzae

The rice blast,caused by fungus Magnaporthe oryzae,is a major constraint to the world food security.Hyphal growth is the foundation of fungal development and proliferation of fungi.To investigate genes involved in hyphal growth of this fungus,digital gene expression tag profiling was used to compare a previously generated temperature-sensitive mutant which defect at hyphae growth and reduction on pathogenicity,with its related wildtype strain.416 genes were detected as differential expression,178 of which were specifically expressed in Guy-11 but down-regulated expression in the mutant.Functional classification analysis revealed the phenotype mutation may be mainly caused by a defection in translational and vacuole- related processes.The results and the protocol used will improve our knowledge on morphogenesis and promote the further study on M.oryzae pathogenesis.     

Research on Arachidonic Acid and Eicosapentaenoic Acid Anabolic Metabolism in Diasporangium sp.

The fatty acids of a strain of Diasporangium sp.had been analyzed by using GC-MS.The fatty acids of twenty mutants were determined.Based on these results,the producing of eicosapentaenoic acid（EPA）supposed via 18∶2,18∶3,20∶3,20∶4 which all belong to ω-6 fatty acids.The ω-3 desaturation was undertaken at arachidonic acid（AA）.In addition,mutant strains resulted in enhanced content of AA which could get two times more than initial strain,but no compact on EPA.     

The Eco-Geographical Distribution of Chemi-cal Composition of Soybean Seeds

The expression of protein and oil content of soybean seeds in worldwide was studied. The results shown that: Latitude and year's weather conditions affected protein content of soybean seeds importantly. Elevation affected oil content significantly. There was important difference in protein / oil content among different eco - geographical regions. The zone, in which there was the highest content of protein / oil, was that one with latitude from 0°to 20°59′ . And in this zone, there were different elevation regions, in which protein or oil content was the highest. For high pro-tein, it was the elevation region with 500 - 1000 m; for oil, it was 0~ 500 m. Protein / oil content was also different among different years. However, the changing range among years in Tropical and Subtropical regions was smaller than that in Temperate Regions. The relationship between protein / oil content and latitude, elevation or soil pH in the regions outside the Tropic of concerned a     

Myristic Acid （MA） Promotes Adipogenic Gene Expression and the Differentiation of Porcine Intramuscular Adipocyte Precursor Cells

Intramuscular fat （IMF） content is considered to be a key factor that affects the marbling, tenderness, juiciness and lfavor of pork. To investigate the effects of myristic acid （MA） on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle （LDM） and treated with 0, 10, 50 or 100μmol L-1 MA. The results showed that MA signiifcantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ（PPARγ） and adipose-related genes, such as glucose transporter 1 （GLUT1）, lipoprotein lipase （LPL）, adipocyte fatty acid binding protein 4 （FABP4/aP2）, fatty acid translocase （FAT）, acetyl-CoA carboxylaseα（ACCα）, adipose triglyceride lipase （ATGL） and fatty acid synthase （FASN）. However, no signiifcant effects of MA were observed on the expression of CAAT enhancer binding protein-α（C/EBPα） or hormone sensitive lipase （HSL）. The expression of pyruvate dehydrogenase kinase 4 （PDK4） was increased by MA during the early stages of differentiation （day 1-3）. In addition, MA also increased the absolute content of C14 （P〈0.001） and saturated fatty acids （SFA） （P〈0.05） to varying degrees, but no effects were observed on other fatty acids. These results suggest that MA might be able to enhance the IMF content of pork and increase the accumulation of myristic and myristoleic acid in muscle, which might have beneifcial implications for human health.     

Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton （Gossypium hirsuturm L.）

Brassinosteroids （BRs） are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene （GhDWF1） involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton （Gossypium hirsutum L.） cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5＇-untranslated region, an ORF of 1 692 bp, and a 120 bp 3＇-untranslated region. The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea, tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD- dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA （day post anthesis） ovule （with fibers） while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules （with fibers） of wildtype （Xuzhou 142） than in ovules of fuzzless-lintless mutant at the same developmental stages （0 and 4 DPA）. The results suggest that the GhDWF1 gene plays a crucial role in fiber development.     

Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression

The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.     

Construction and Virulence of Filamentous Hemagglutinin Protein B1 Mutant of Pasteurella multocida in Chickens

Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two filamentous hemagglutinin genes, fhaB1 and JhaB2, are the potential virulence factors. In this study, an inactivationfhaB1 mutant ofP. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of thefhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation offhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. ThefhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90% convalescent chicken serum. ThefhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum （P〈0.007）. These results confirmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.     

The Twin-Arginine Translocation （Tat） Pathway Is Essential for Virulence,Flagellation, and Chemotaxis in Xanthomonas oryzae pv. oryzicola Strain RsGD42

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice （Oryza sativa）. Genetic determinants （tatABC genes） of the twin-arginine translocation （Tat） pathway from X. oryzae pv. oryzicola strain RsGD42 were cloned and characterized, meanwhile, a tatC disruption mutant was generated. The tatC mutant lacked detectable flagella and was highly impaired in motility and chemotaxis. Furthermore, it was observed that the tatC mutant exhibited a reduced production of extracellular polysaccharide （EPS） and a significant reduction of virulence on adult rice plants compared to wild type strain. However, the tatC mutation in X. oryzae pv. oryzieola strain RsGD42 did not affect the growth rate and the ability to induce hypersensitive response （HR） in nonhost tobacco （Nicotiana tabacum L. cv. Samsun）. In conclusion, the data indicated that the Tat pathway significantly contributed to the virulence of X. oryzae pv. oryzicola.     

Cultivar and Environmental Variation of β-glucan Content in Chinese Barleys

β-glucan is a polysaccharide compound closely related to the quality of barley used as malting,feed and food. Low β-glucan content is expected for brewing and feed barley, while high β-glucan content is desirable for food barley. The β-glucan content of barley genotypes collected from various areas of China as well as from Canada and Australia were assayed. Meanwhile a multi-locations trial was conducted to determineβ-glucan content of 10 barley cultivars in 8 locations for two successive planting years. The results showed that barley genotypes from Tibet and Xinjiang had higher β-glucan content and the genotypes with higher than 8%of β-glucan content were detected in Tibet barleys, being valuable for use in the development of healthy food.Barley cultivars being planted now in winter-sowing areas of China had basically the same β-glucan content as those from Canada and Australia. Barley seeds produced in Hangzhou had lower β-glucan content than seeds from the original areas. There was a highly significant difference in β-glucan content among 10 barleys, 8locations and between years. On an average of two years, Xiumei 3 and Kongpei 1 had the highest and lowestβ-glucan content, respectively, and Taian and Hangzhou produced the highest and lowest β-glucan content barley seeds, respectively. Analysis of AMMI model showed that interaction effect between cultivar and environment was highly significant in both experimental years, and was dependent on cuitivar, suggesting that it is important to plant the suitable cultivars in a particular area in order to obtain barley seeds with reasonableβ-glucan content.     

Identification of a Long Rice Spikelet Mutant

A spontaneously occurring rice (Oryza sativa L.) mutant, characterized by homeotic conversion in glumes and stamens, was found in the progeny of a cross. The mutant showed long glumes and glumaceous lodicules and morphological transformation of stamens into pistils. Mutant florets consisted of 1 to 3 completely developed pistils, some pistilloid stamens with filaments, but tipped by bulged tissue and 0 to 3 stigmas. It seems that the mutant phenotype of the homeotic conversions in glumes and stamens is similar to that of the B loss-of-function mutants in Arabidopsis and Antirrhinum. The mutant is controlled by a single recessive gene as a segregation ratio of 3:1 (wild type to mutant plants) was observed in the F2 generation.     

Purification of Aminopeptidase N Protein and Differences in cDNAs Encoding APN1 Between Susceptible and Resistant Helicoverpa armigera Strains to Bacillus thuringiensis Toxins

The brush border membrane vesicles (BBMVs) in midgut of Helicoverpa armigera were successfully separated, and most of the Aminopeptidase N (APN) activities in BBMV were preserved. The 3-[(3-chlor-amidopropyl) dimethylammonio]-l-propane-sulphonate (CHAPS) can enhance the dissolution of BBMV, and phosphatidylinositol-specific phosopholipase C (PI-PLC) can cleave the APN from midgut membrane. The APN was primarily purified using a Mono-Q column. The results of immunoblotting showed that the 120 and 170 kDa proteins in the BBMV could bind CrylAc, and 120 kDa APN was a glycosylphosphalidylinositol(GPI)-anchored protein. Two Bt-resistant strains (Bt-P, Bt-M) were obtained after being selected for more than five years in laboratory using Bt insecticides and Bt transgenic cotton incorporated into diet separately. The resistance of Bt-P and Bt-M were 1 083.3 and 48.7 times that of susceptible strain. The genes encoding APNI in midgut of susceptible and resistant H.armigera were cloned by PCR and RACE techniques. The inferred amino acid sequences of APNI possessed the common character of APN family in insects. In comparison with APNI in susceptible strain, three nucleotide mutations were observed in the APNI of Bt-M strain and resulted in two amino acid replace in the putative protein sequences, and eight nucleotide mutations were observed in Bt-P strain and resulted in five amino acid replace.     

高产谷胱甘肽酵母菌株的筛选及其抗乙硫氨酸诱变研究(英文)

[Objective] The aim of this study was to screen Saccharomyces for glutathione over-production. [Method] Ethionine-resistant mutants were obtained through UV mutagenesis and rational screening. [Result] A high GSH-producing strain HSJB1 was isolated from soil, and the biomass for this strain by flask shaking fermentation was 3.87 g/L while the GSH yield was 91.87 mg/L. According to the morphological, physiological and biochemical characteristics of cells, this strain was primarily identified as Saccharomyces cerevisiae. An ethionine-resistant mutant YBS77 was obtained through UV mutagenesis of the original strain HSJB1, and the biomass for this strain by flask shaking fermentation was 7.60 g dry cell weight/L while the GSH yield was 211.96 mg/L. [Conclusion] The biomass of the mutant obtained by breeding is increased by 96.38% than that of the original strain, and the GSH yield of the mutant obtained by breeding is increased by 130.72% than that from the original strain, which indicates that the breeding method is feasible.     

Phytic Acid Contents of Wheat Flours from Different Mill Streams

The phytic acid contents of eight fractions of wheat flours from different mill streams and those in wheat brans, which were separated by different sieves into various sizes, were determined and analyzed. A rapid method for phytic acid assay by adding thioglycolic acid （mercapto acetic acid） with 2,2-bipyridine was used, with an acidic iron-Ⅲ-solution of known iron content. The amount ofphytate was indicated by the decrease in iron in the supernatant. Significant differences were observed in phytic acid content among different milling streams and different cultivars （P〈0.05）. On an average, coarse bran had the highest phytic acid content （53.85 mg g^-1）, and the shorts had 28.48 mg g^-1. The B5 break flour had a higher phytic acid content （4.8 mg g^-1） than the B7 （2.75 mg g^-1） and B8 （2.03 mg g^-1） reduction flours. Lower values were found in the B3, B6 and B7 flours （1.07, 0.79, and 0.76 mg g^-1, respectively）. The phytic acid contents of bran decreased with smaller bran sizes, ranging from 54 to 5.09 mg g^-1.     

Effects of Crop Rootzone Non-Pressure Subirrigation on Tomato Physiological Characteristics, Yield, and Quality

The purpose of this article is to study the effects of different water elevations of non-pressure subirrigation on some indexes of tomato, including soil water status around crop rootzone, morphological indexes, physiological indexes, photosynthetic indexes, yield, quality, and water use efficiency. With the tomato materials of Dongsheng 1, the irrigation experiment was carried out in the greenhouse, and significance analysis was done on the experiment data through the software of DPS. The results showed that different water elevations, had significant influence on the growth, yield, and quality of tomato. The yield of the 6-cm treatment was the highest, the 3-cm treatment was inferior to the 6-cm treatment, and the 0-cm treatment was the lowest. However, the WUE was 0 cm 〉 3 cm 〉 6 cm. The sugar/acid and soluble protein was the highest under the 0-cm treatment, and the content of ascorbic acid did not decrease considerably. When compared to the 0-cm treatment, the ascorbic acid content of 6-cm and 3-cm treatment increased by 19.2 and 6.8%, respectively. These irrigation methods can satisfy the requirements of tomato growth; different water elevations have different influences on the tomato soil water status around crop rootzone, the physiological characteristics, and yield. It also harmonized the percentage between sugar and acid, increased the content of soluble protein and ascorbic acid, and made tomato more delicious. The irrigation methods can improve the quality of tomato by water control, which is worth promoting in the agricultural production.     

Comparison of Acetylcholinesterase Characteristics Between Resistant and Susceptible Strains of Helicoverpa armigera （Hubner）

The sensitivity of a susceptible and two resistant strains of cotton bollworm, Helicoverpa armigera, to phoxim, malathion and methomyl was determined by a topical application of bioassay method. YG strain, collected from field of Yanggu, Shandong Province of China, possessed 7-, 13- and 20-fold of resistance to the above three antiacetylcholinesterases based on the comparison of LD50 values with a laboratory susceptible strain. There were not significant difference of the specific activity and the Vmax value among the three strains. But the affinity of AChE tO acetylthiocholine （ATCh）, in YG strain was the lowest among the three strains tested. A cDNA encoding partial AChE gene was cloned from the three strains by RT-PCR and there was one nucleotide acid difference between YG strain and other two strains which resulted in no amino acid mutation. This partial AChE gene was used as a probe to perform Southern blot. The results indicated that there was no gene amplification in resistant cotton bollworm. Altered AChE with a decreased sensitivity to inhibitors appeared to be one of important resistance mechanisms in cotton bollworm against OP and carbamate compounds.     

Comparing Genetic Characteristics of Retrotransposon TOS17 During Different Tissue Culture Processes in the Rice Cultivars Nipponbare and Shishoubaimao

As a retrotransposon, TOS17 was a useful tool for rice genetic and functional genomic research. To ascertain the feasibility of constructing a TOS17 insertion mutation library in the rice cultivar Shishoubaimao, the genetic and expression characteristics of TOS17 were analyzed. We made solid and suspension tissue cultures and confirmed the copy numbers of TOS17 at different time points in both tissue culture processes by real-time quantitative PCR （RT-qPCR）. Three primary copies of TOS17 were detected in naturally grown Shishoubaimao. TOS17 was activated by tissue culture, and the copy numbers of TOSI7 increased along with a prolonged tissue culture time in both the Nipponbare and the Shishoubaimao cultivars. Therefore, Shishoubaimao is a potential candidate for constructing a TOS17 insertion mutant library. Compared with Nipponbare, TOS17 was more active in Shishoubaimao during tissue culture. Higher copy numbers of TOS17 were obtained with the suspension tissue culture process than with the solid tissue culture process over the same time courses. We concluded that 3-4 months of suspension tissue culture time is suitable for constructing a TOS17 insertion mutant library in Shishoubaimao.     

Effect of Low Content Chlorophyll on Distribution Properties of Absorbed Light Energy in Leaves of Mutant Rice

This paper reported the diurnal variations of photochemical efficiency of PSII, thermal dissipation rate and other physiology process in the low content chlorophyll mutant rice and its wild type under field conditions, and analyzed the difference of absorbed light distribution between the two rice varieties in a day. The results showed that the mutant had poor absorbed light because of its little light absorption coefficient, but higher electron transportg rate could partly reduce the disadvantageous effect of deficient absorbed light in mutant. Compared with wild-type rice, the mutant had less excess excitation energy and the fraction of absorbed light allocated to photochemical process was more.