Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation.

The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica papaya L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). The Cu/Zn SOD was expressed in Escherichia coli and purified by His-tag technique. Two active forms of the enzyme (30% dimer and 70% monomer) in equilibrium were observed. The activity of the dimeric enzyme was higher than that of the monomeric form. The thermal inactivation rate constant K(d) values calculated for the dimer and monomer at 90 degrees C were -0.0203 and -0.0216 min(-1), and the half-lives for inactivation were 41.9 and 31.8 min, respectively. This indicated that the dimeric enzyme was more stable than its monomeric form. The dimerization of the enzyme was inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it was not affected under alkaline pH (above 9.0). Both activity and forms of the enzyme were not affected by 1-4% SDS. Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin or chymotrypsin. In addition, mutation of the papaya Cu/Zn SOD at position 48 from Leu to Phe (L48F) affected the association of monomer, whereas a mutant with Lys substitution (L48K) at the same position tended to dissociate into monomeric form.     

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n = 17) and green Texas cichlid (Herichthys cyanoguttatus) (n = 1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed‐field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%–100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 10³ CFU/fish and 2.08 × 10² CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.     

Conversion of Squid Pen to Homogentisic Acid via Paenibacillus sp. TKU036 and the Antioxidant and Anti-Inflammatory Activities of Homogentisic Acid

The culture supernatant of Paenibacillus sp. TKU036, a bacterium isolated from Taiwanese soils, showed high antioxidant activity (85%) when cultured in a squid pen powder (SPP)-containing medium at 37 °C for three days. Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) was isolated and found to be the major antioxidant in the culture supernatant of the SPP-containing medium fermented by Paenibacillus sp. TKU036. Tryptophan was also present in the culture supernatant. The results of high-performance liquid chromatography (HPLC) fingerprinting showed that HGA and tryptophan were produced via fermentation but did not pre-exist in the unfermented SPP-containing medium. Neither HGA nor tryptophan was found in the culture supernatants obtained from the fermentation of nutrient broth or other chitinous material, i.e., medium containing shrimp head powder, by Paenibacillus sp. TKU036. The production of HGA via microorganisms has rarely been reported. In this study, we found that squid pen was a potential carbon and nitrogen source for Paenibacillus sp. Tryptophan (105 mg/L) and HGA (60 mg/L) were recovered from the culture supernatant. The isolated HGA was found to have higher antioxidant activity (IC₅₀ = 6.9 μg/mL) than α-tocopherol (IC₅₀ = 17.6 μg/mL). The anti-inflammatory activity of the isolated HGA (IC₅₀ = 10.14 μg/mL) was lower than that of quercetin (IC₅₀ = 1.14 μg/mL). As a result, squid pen, a fishery processing byproduct, is a valuable material for the production of tryptophan and the antioxidant and anti-inflammatory HGA via microbial conversion.     

拟南芥AAP1基因的克隆及植物表达载体的构建

拟南芥氨基酸透性酶1(AAP1,也叫NAT1)是植物中第一个被分离的氨基酸转运蛋白,在拟南芥主根、侧根、根毛及根表皮细胞中均有增加氨基酸含量的作用。本研究利用RT-PCR的方法,从拟南芥中克隆了该基因,测序结果与GenBank(NM-104616.4)中已发表的拟南芥AAP1(AtAAP1)基因的同源性高达99.93%;并将AtAAP1基因与pCAMBIA3300构建p3300-AAP1植物表达载体,为过表达AtAAP1基因,提高作物氨基酸含量,从而改善作物氮素利用效率提供参考。     

高产田不同玉米品种养分吸收、分配及利用效应研究

以玉米高产为目标,采用大田试验方法,研究了高产管理措施下不同玉米品种对N、P、K养分的吸收、分配和利用效应。结果表明:随玉米生育进程,不同玉米品种N、P、K养分含量呈缓慢下降趋势,植株体内的养分阶段净累积量呈双峰或单峰曲线,养分累积量大小顺序为N>K>P;从全生育期来看,"丰玉4号"吸收N、K量最高,分别为275.73 kg/hm2和243.55 kg/hm2,而"京单28"吸P量最高,为50.28 kg/hm2;各器官养分分配比例随生育时期变化不大;产量以"丰玉4号"最高,为12 391.05 kg/hm2,每形成100 kg子粒所需要的氮钾养分量均以"京单28"最高,"丰玉4号"次之,"浚单20"最低;对磷的需要量仍以"京单28"最高,但"丰玉4号"和"浚单20"2品种基本相当。3品种对N、P2O5、K2O的吸收比例为1∶0.40~0.44∶0.98~1.06。     

植物生长调节剂对库尔勒香梨产量与品质的影响

为了明确植物生长调节剂在库尔勒香梨(Pyrus brestschneideri Rehd.)生产中的施用效果,该田间试验以20 a树龄库尔勒香梨为研究试材,研究植物生长调节剂PBO(增效醚,包含细胞分裂素BA与生长素衍生物ORE)、PP333(15%多效唑)和复硝酚钠对库尔勒香梨果实产量及内外部品质的影响,分别设置不同生长调节剂、不同浓度、清水及空白喷施处理,共计17个处理,每处理3次重复。结果表明:喷施植物生长调节剂可以显著提高库尔勒香梨产量,可以降低果皮厚度、果实硬度、果实总酸含量,还可以提高果实脱萼率、红晕果率、果实还原糖含量和维生素C含量,其中以喷施PBO 300倍液效果最佳。与对照处理相比,喷施PBO 300倍液处理的库尔勒香梨产量、红晕果率、脱萼果率及果实还原糖含量分别提高了63.41%、42.01%、38.97%和22.22%,而果皮厚度、果实硬度及果实总酸含量分别降低了26.44%、13.42%和31.3%。     

新城疫病毒F基因在新型杆状病毒表达系统中的表达及重组病毒的免疫原性

将新城疫病毒（四平株）F基因插入到pFastBac Ⅰ质粒中，构建了重组质粒pFF。pFF转化大肠杆菌DH10Bac后，F基因在助手质粒（helper plasmid)提供转座酶的情况下，通过转座进入Bacmid，构建了重组Bacmid(re-Bacmid)。在含有X－gal/IPTG、庆大霉素、卡那霉素和四环素的琼脂平板上，筛选白色菌落后，提取re-Bacmid转染Sf-9昆虫细胞。在昆虫细胞内，re-Bacmid经复制、表达、装配、形成重组杆状病毒，并表达F蛋白。经SDS－PAGE和Western-blot分析，表达的F蛋白的分子大小为63000，并证明在昆虫细胞内表达的蛋白能部分糖基化。表达的F蛋白约占细胞总蛋白的10％。动物试验证明，约建的重组杆状病毒具有良好的免疫原性。     

Subchronic toxicity test for two thermotolerant filamentous fungi used for single cell protein production.

Safety evaluations of two thermotolerant filamentous fungi, Cephalosporium eichhorniae 152 (C. 152) and Rhizopus chinensis 180 (R. 180), grown on a sugar-salts medium were carried out through feeding the biomases to rats at 20% or 40% dietary levels for 90 days. There was a control group fed soybean meal. Weight gain and feed consumption for rats fed 20% C. 152 were equal to those for the control animals, but were depressed in the other three groups, especially the rats fed R. 180. All animals appeared normal and healthy except that transient alopecia was found for a short duration in the fungi-treated rats in the initial period. The cause of this lesion is not clear. At the end of the feeding trial, clinical determinations of constituents in blood and urine samples were conducted. The animals were autopsied and weights for four organs were taken. Histopathological examinations for 26 different tissues were carried out. Mild changes were found in both C. 152 and R. 180-treated rats but most of the these were not considered to be related to treatment.