大肠杆菌yqhD基因的克隆与表达

以大肠杆菌E scherich ia coli K-12基因组DNA为模板,利用PCR技术扩增得到假定的氧化还原酶(pu tative ox idoreductase)基因yqhD,将它连接到克隆质粒pGEM-3zf(+)上,得到重组质粒pGEM-yqhD,对此重组质粒进行序列测定,对其DNA序列分析表明,yqhD基因全长为1 164 bp。再将yqhD基因插入表达载体pSE 380,构建成重组子pSE 380-yqhD,并在E.coli BL 21中获得表达。研究表明,以1,3-丙二醇为底物时,基因工程菌在30°C下,以1.0 mm o l/L IPTG诱导12 h的酶活力达到3.13 U/mL,比对照菌株提高4.4倍。     

含有His 5基因用于酵母人工染色体重组筛选的置换型载体的构建

人工酵母染色体(YAC)可以克隆和分析大片段的染色体DNA,并且可以分离那些在大肠杆菌(Escherichia coli)中不可能得到的序列。实验将His5基因插入到一个Ura3基因的中间,构建了一个新的质粒pLRH33,从而打断了Ura3基因使之不能表达。利用His5基因两端各留下的部分Ura3基因片段,同源重组到YAC臂上原有的Ura3基因中,使重组后的YAC的标记性状从Rra^ His^-变成了His^ Ura^ 。用质粒pLRH33的5．5kb线性片段以一定比例和需要重组到YAC上的目的基因pBAC300的50kb片段相混合,同时作酵母菌转化,在His^-Ura^ 培养基上得到大量带His5基因的阳性克隆。在检测的1200个克隆中,有250个目的基因阳性克隆,占受检克隆的22．5％。表明质粒pLRH33可以有效地用作YAC重组的筛选标记：     

表达伪狂犬病毒gC糖蛋白基因重组腺病毒的构建及其免疫效果

通过双酶切将伪狂犬病毒(Pseudorabies virus,PRV)Fa株gC囊膜糖蛋白基因片段亚克隆到5型腺病毒AdMax系统穿梭质粒pDC316中,得到pDC316-gC。用此质粒与腺病毒DNA辅助质粒pBHGloxΔ E1,3Cre共转染293细胞,包装出重组腺病毒Adv-gC。通过PCR鉴定和病毒空斑纯化,得到纯的高效价Adv-gC病毒液。间接免疫荧光实验证明此重组病毒能表达PRV gC蛋白。该病毒经肌注免疫Balb/c小鼠,能诱导小鼠产生特异的体液和细胞免疫反应。攻毒试验表明,此重组病毒能提供100%的免疫保护。     

睾丸酮丛毛单胞菌活化因子基因的质粒载体构建及表达

提取睾丸酮丛毛单胞菌(Comamonas testosteroni)的基因组DNA，利用PCR扩增activator基因，将扩增产物克隆到pKtac1(含强启动子tac)中，构建重组质粒pKtac1-act1和 pKtac1-act2。经酶切分析和测序，鉴定出正确的重组质粒pKtac1-act1和 pKtac1-act2。将重组质粒转化入大肠杆菌(Escherichia coli)HB101中，用酶联免疫分析方法测定细菌总蛋白质中的activator表达量；将pAX1(含3α-HSD/CR基因)分别和重组质粒(pKtac1-act1和pKtac1-act2)一起转化入E. coli HB101中，测定细菌总蛋白质中的3α-HSD/CR和activator的表达量。结果表明：重组质粒能明显提高activator的表达；pAX1与重组质粒共转化的蛋白质粗提物中，activator和3α-HSD/CR的表达量都显著提高。     

农杆菌介导的蓝色基因转化中国水仙

从矮牵牛(Petunia hybrida)花瓣中分离得到编码蓝色基因F3'5'H(类黄酮3',5'羟基化酶)的Hf2基因(1527 bp),从牵牛(Ipomoea nil)花瓣中分离得到dfr基因(二氢黄酮醇4-还原酶,1212 bp),将正向Hf2片段和反向dfr片段连接到pBRLys双元载体质粒上,得到具有Ubi(ubiquitin)组成型启动子的植物表达载体pU-bi-Hf2-dfr.利用冻融法将双价重组质粒pUbi-Hf2一afr导入农杆菌(Agrobacterium tumefaciens)菌株EHA105中,菌落PCR鉴定农杆菌转化子.以中国水仙(Narcissus tazetta var.chinensis)愈伤组织为材料,进行遗传转化.CTAB法提取130株中国水仙遗传转化再生苗的基因组DNA,进行PCR和PCR-Southem blot检测,获得3株阳性转基因植株.     

不同-10区保守序列的tac启动子表达睾丸酮丛毛单胞菌中的活化因子

提取睾丸酮丛毛单胞菌（Comamonas testosteroni）的基因组DNA,利用PCR扩增活化因子（activator）基因.将扩增产物克隆到pKtac1（含强启动子tac）中,构建含全长activator基因（564bp）的重组质粒pKtac1-act1和含部分activator基因（409bp）的pKtac1-act2.测序结果发现重组质粒pKtac1-act2的tac启动子-10保守区由TATAAT突变为TATGTT.将pKtac1-act1和pKtac1-act2分别进行酶切和连接,获得重组质粒pKtac1-act3（含启动子-10保守区为TATAAT,409 bp的部分activator基因）和pKtac1-act4（含启动子-10保守区为TATGTT,564 bp的全长activator基因）.将4个重组质粒分别转化入宿主菌Escherichia coli HB101中,用酶联免疫分析方法测定细菌总蛋白质中的activator表达量;将pAX1（含3α-hsd/CR基因）分别和4个重组质粒一起转化入宿主菌E.coliHB101中,测定细菌总蛋白质中的3α-hsd/CR和activator的表达量.结果表明：启动子-10保守区为TATAAT的质粒比-10保守区为TATGTT的质粒有更高的转录活性;过量的actvitor表达可能影响质粒pKtac1-act3表达的稳定性;启动子-10区保守序列的下降突变（pKtac1-act2,TATAAT→TATGTT）及tac启动子的丢失（pKtac1-act3,继代培养4次）揭示了宿主菌E. coli HB101的自身防护机制.     

黑曲霉木聚糖酶基因（xynB）的克隆及真核分泌表达

通过RT-PCR方法,以黑曲霉(Aspergillus niger)GIM3.452总RNA为模板,克隆出木聚糖酶B(xy-lanase B,xynB)基因的成熟肽编码序列(567 bp),编码188个氨基酸.将其与猪腮腺分泌蛋白(parotid secretoryprotein,PSP)基因的信号肽序列通过重叠延伸PCR(SOE-PCR)得到拼接片段PSxynB,并将其克隆到真核表达载体pcDNA6/HisTMA中,得到重组质粒pcDNA-PSxynB,重组质粒经过酶切、测序鉴定,证实含有目的片段,且构建正确.在脂质体介导下将重组质粒pcDNA-PSxynB转染猪肾细胞(PK15),通过RT-PCR证实其在PK15细胞中表达,并在细胞培养液中测到了木聚糖酶活最高达36.4 IU/mL.     

睾丸酮丛毛单胞菌teiR基因载体构建及在大肠杆菌中的表达

提取睾丸酮丛毛单胞菌(Comamonas testosteroni)基因组DNA,利用PCR扩增teiR基因,将扩增产物克隆到pKtac2(含强启动子tac)和pK18中,构建重组质粒pKtac2-teiR和pKteiR100。将重组质粒分别转化大肠杆菌(Escherichia coli)HB101, 提取细菌总蛋白,ELISA测定细菌总蛋白中teiR基因的表达量,结果表明: 含tac强启动子的pKtac2-teiR重组质粒具有更高的转录活性;将p6(含3a-HSD/CR基因)分别和重组质粒(pKtac2-teiR和pKteiR100)共转化大肠杆菌(Escherichia coli)HB101, 测定细菌总蛋白中3a-HSD/CR 和teiR基因的表达量,结果表明: 在大肠杆菌中teiR可以明显促进3a-HSD/CR 的表达, pKtac2-teiR与p6共转化后teiR基因的表达量比pKteiR100与p6共转化后teiR基因的表达量高,但是pKtac2-teiR与p6共转化后的3a-HSD/CR的表达量低于pKteiR100与p6共转化后的3a-HSD/CR的表达量。     

牛nanog基原核表达载体构建及其在大肠杆菌中的表达

从6周龄的胎牛(Bos taurus)原始生殖嵴中提取总RNA,通过RT-PCR扩增nanog基因,将其克隆到PMD-18T载体,然后再亚克隆到pGEX-KG表达载体上,获得原核表达质粒pGEx-KG-nanog,限制性内切酶分析和DNA测序证明所插入片段为牛nanog基因编码序列.重组质粒转化大肠杆菌JM109(Escherichia coli),在不同的培养温度(25、30和37℃)和不同浓度的IPTG(0.10、0.25、0.50、1.00和2.00mmol/L)诱导下均获得了表达,结果表明,培养温度和IPTG浓度对GST-Nanog融合蛋白在大肠杆菌中的表达影响甚微;经Western blot检测证实该蛋白约60kD,并具有GST抗原活性,证实目的蛋白为Nanog蛋白.     

猪繁殖与呼吸综合征病毒（PRRSV） BJ-4株ORF2基因的原核表达

通过PCR方法从重组质粒pGEM-ORF2扩增得到缺失N端疏水序列的基因片段dORF2(deleting ORF2)。将dORF2克隆至原核高效表达载体pGEX-4T-2，在大肠杆菌(Escherichia coli)BL21细胞中成功地表达了猪繁殖与呼吸综合征病毒(PRRSV)重组蛋白GST-dORF2，表达量为22％。Westem-blot结果表明重组蛋白可被谷胱甘肽S-转移酶(GST)抗体识别。表达的重组蛋白为进一步研究PRRSV次要结构蛋白的结构和功能奠定了基础。     

Soil-profile distribution of primary and secondary plant-available nutrients under conventional and no tillage

Nutrient distributions under no tillage (NT) compared with conventional disk-and-bed tillage (CT) management in the warm, humid region of the southeastern USA need to be assessed so that future placement, quantity, and type of fertilizers can be altered, if necessary, to efficiently match crop demands. We determined soil-profile distributions of pH, N, P, S, K, Ca, Mg, Na, Zn, Fe, Mn, and Cu to a depth of 0.9 m at the end of 8.5 years of continuous CT and NT management on a Weswood silty clay loam (fine, mixed, thermic Fluventic Ustochrept) in southcentral Texas. Most dramatic changes occurred within the 0–0.05 m depth, where soil under NT had lower pH, Fe, and Cu than under CT, but greater P, K, Zn, and Mn. Greater P and K under NT than under CT also occurred below the till-zone (0.15–0.3 m). At a depth of 0–0.3 m, soil under NT contained greater amounts of extractable P, K, Zn, Fe, Mn, and Cu than under CT. Nitrogen fertilization had little effect on nutrient distributions, except resulting in greater extractable K at 0–0.05 m and greater nitrate at 0–0.15 m. Few changes in soil-profile distributions were observed for extractable S, Ca, Mg, and Na. Long-term continuous use of NT on this fine-textured, high-fertility (except for N) soil had no apparent adverse effects on nutrient distributions relative to CT, but enhanced conservation and availability of P, K, Zn, Fe, Mn, and Cu near the soil surface where crop roots proliferate.     

Assessing the effectiveness of conservation measures: Resolving the “wicked” problem of the Steller sea lion

“Wicked” problems are those that are complex and that change when solutions are applied. Many conflicts in conservation fall into this category. The study approached the problem of how to constrain the apparent wickedness of a problem in the conservation management of a species by using simple empirical indicators to carry out iterative assessment of the risk to a population and to document how this risk evolves in relation to the addition of new data and the implementation of management actions. Effects of high levels of uncertainty within data and also concerning population structure were examined through stochastic simulation and by exploration of scenarios. Historical trends in the example used, the Steller sea lion, showed rapid declines in abundance in some regions during the 1980s. The current total population is 130,000-150,000 Steller sea lions through Alaska and British Columbia and this number has been stable since about 1990 in spite of regional differences in population dynamics. Regional differences in the sequence of changes in the number of pups and non-pups, suggested that an internal re-distribution of juveniles could have happened between 1980 and 1990. Current productivity also appears close to the long-term mean. Stochastic population projection using various scenarios showed that, based upon this history, the risk of extinction for the population has declined and is below reasonable thresholds for considering the population to be endangered. The trends in risk suggest that management actions taken since 1990 have probably been effective. Consequently, the conservation management objectives for the Steller sea lion are probably being met. The approach provides a mechanism, based upon experience and scenario analysis, for exploring future policy options and may help to constrain the debate amongst stakeholders about the cost-benefit trade-offs associated with different options.     

Enhanced establishment and growth of giant cardon cactus in an eroded field in the Sonoran Desert using native legume trees as nurse plants aided by plant growth-promoting microorganisms and compost

To evaluate the feasibility of long-term desert reforestation technology of mixed vegetation, cardon cactus (Pachycereus pringlei) seedlings from indoor and outdoor nurseries were planted in the field adjacent to one seedling of potential legume nurse trees: mesquite amargo (Prosopis articulata), yellow palo verde (Parkinsonia microphylla), and blue palo verde (Parkinsonia florida). Some of the planting holes were also supplemented with common dairy compost. Additionally, the combinations of legume tree–cactus were inoculated with either a consortium of desert arbuscular mycorrhizal (AM) fungi, plant growth promoting bacteria (PGPB; the diazotroph Azospirillum brasilense Cd, and the phosphate solubilizer Paenibacillus sp.), or a mixture of all. The field experiments were evaluated periodically during 30 months for survival and growth. Cardons reared in an outdoor screen house survived better in the field than those reared in a controlled growth chamber and hardened later outdoors. Association with any legume nurse tree increased survival and enhanced growth of untreated cardons. For cardons growing alone, application of either compost, AM fungi, and all the treatments combined increased survival. For these plants, no treatment affected plant growth during the first 3 months after transplanting. Later, all treatments, except for AM fungi, enhanced plant growth. However, only 2 years after transplanting the enhanced growth effect of AM fungi was also significant. In the presence of the legume nurse trees, transient positive effects on cardon growth were recorded. General evaluation after 30 months of cultivation showed that the treatments positively affected cardon growth when growing alone or in combination only with mesquite amargo but not with the other two legume trees. This study proposes that young legume trees have the capacity to enhance survival and growth of cardon cactus, depending on the legume cactus combination. Additional treatments such as compost or PGPB can either amplify the effect or else attenuate it.     

Karyotypic and genome size variation in Cajanus cajan (L.) Millsp. (pigeonpea) and some wild relatives

Detailed karyotypes and 4C DNA amounts have been studied in five cultivars of Cajanus cajan and 20 species belonging to Cajanus, Rhynchosia, Dunbaria, Flemingia and Paracalyx. C. cajan shows intraspecific variability and its karyotype is most similar to that of C. cajanifolius (sect. Cajanus) in the morphology and number of satellite chromosomes and the lack of any correlation between chromosome size within the complement and asymmetry. Karyotypes of C. lineatus and C. sericeus belonging to sect. Atylia are similar with respect to maximum r-index and the ratio of longest and shortest chromosomes in their respective complements. C. acutifolius (sect. Frutocosa) is distinct in having a very low ratio (1.44) between the longest and the shortest chromosomes, while in C. albicans, C. goensis, C. scarabaeoides (sect. Cantharospermum) there is no chromosome pair with r-index >2.0. C. mollis and C. volubilis (sect. Volubilis) show similarity with regard to ratio between the longest and the shortest chromosomes and a distinct chromosome pair with an arm ratio of 2.7 and C. platycarpus is distinct (sect. Rhynchosoides) in having the smallest ratio (1.36) between the longest and the shortest chromosomes. 4C DNA amounts in Cajanus and Rhynchosia vary between 3.28 pg to 11.69 pg and 4.92 pg to 11.13 pg respectively, while in Dunbaria, Flemingia and Paracalyx these vary between 4.70 and 7:06 pg. In the genus Cajanus karyotypic features and 4C DNA amounts agree with the sectional classification and bring out a close relationship between C. cajan and C. cajanifolius. This is supported by studies based on seed protein patterns, isozyme analysis, trypsin and chymotrypsin inhibitor patterns, RFLP and RAPD data and crossability relationships. A clear relation between DNA amount and abnormalities effecting the extent of pairing and recombination in interspecific hybrids shows the importance of this study in developing future breeding programme involving C. cajan and its wild relatives that are of potential value.     

Effect of some fungicide seed treatments on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybean [Glycine max. (L) Merr.]

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation.     

Einfluss der düngungsmethoden auf die entwicklung von zuckerrübenkrankheiten

Während neunjähriger (1994-2002) Untersuchungen in der Versuchsanstalt Ba?cyny (Fahlerde, Fall-Gleyboden, ausgebildet aus leichtem Lehmboden) wurde der Befall mit Krankheiten an der Zuckerrübe in Abhängigkeit von der Düngungsmethode bewertet. Folgende Methoden wurden angewendet: A - Mineraldünger, B - Stalldung und Mineraldünger, C - Stroh-, Gründung und Mineraldünger, D - Kompostdünger und Biopräparate (ökologische Düngung). Auf den Objekten A, B, C wurden bei konstanter Phosphor-Kalium-Düngung die Stickstoffgaben von 0 bis 180 kg.ha^?1 differenziert. Auf dem ökologischen Objekt D ohne Mineraldünger wurden statt Stickstoff die Biopräparate P₅₀₀ und P₅₀₁ verabreicht. Der geringste Befall von Krankheiten wurde an der Zuckerrübe auf jenem Objekt festgestellt, das mit Kompost und den Biopräparaten gedüngt worden war. Im Rahmen der konventionellen Düngungsmethoden wurde allgemein die höchste Intensität der Krankheiten an der mit Stroh, Gründung und Mineraldünger gedüngten Zuckerrübe beobachtet. Ausnahmen von dieser Regel bildete das Vorkommen von echtem Mehltau und Keimlingsfäule. Auf den Objekten A, B, C nahm die Intensität der Krankheiten mit der Steigerung der Stickstoffgabe zu. Auch die auf Objekt D verabreichten Biopräparate stimulierten die Entwicklung der Krankheiten. The effect of various fertilization methods on the diseases of sugar beet was estimated on the basis of nine-year studies (1994-2002) conducted in the Experimental Production Farm in Ba?cyny on loess-based, sedimentary-gley, light-clay soil. The following fertilization methods were applied: A - mineral fertilization, B - mineral and manure fertilization, C - straw, green manure and mineral fertilization, D - ecological compost and biodynamic preparations fertilization. In the objects A, B and C fertilized with phosphorus and potassium, various doses of nitrogen ranging from 0 to 180 kg/ha^?1 were used. In the ecological object D, where mineral fertilization was not used, biodynamic preparations P₅₀₀ and P₅₀₁ were applied instead of nitrogen. The lowest ratio of the diseases of sugar beet was observed in the object fertilized with the compost and biodynamic preparations. In view of the conventional fertilization methods, generally the highest incidence of diseases was noted in the sugar beet fertilized with straw, green manure and mineral fertilizers. The exception was the occurrence of powdery mildew and the black leg. In objects A, B and C the incidence of the diseases increased with the increase in nitrogen doses. The applied biodynamical preparations in the object D stimulated the development of diseases too.     

Prospects for control of Pythium damping-off of lettuce with Trichoderma,Gliocladium and Enterobacter spp.

Summary Damping-off of lettuce (Lactuca sativa) caused by Pythium ultimum was studied in pots containing a non-sterile potting mix in the glasshouse. Fifty P. ultimum sporangia g^-1 compost reduced the plant stand to 15% and shoot dry weight to 18%, but this reduction was totally prevented by applying Trichoderma harzianum at 2×10⁵ viable propagules g^-1 potting mix. Gliocladium virens and Enterobacter cloacae also alleviated the damping-off. E. cloacae did not adversely affect the action of the fungal antagonists. The co-existence of the bacterial and fungal antagonists was revealed on the root surface and inner surface of the testa by scanning electron microscopy, indicating the compatibility of the biocontrol agents.     

细菌、真菌及植物氮营养信号研究进展

氮素吸收代谢是所有生物生命活动的核心部分,因此,各种生物对氮的吸收与同化过程都有精细的调节。这种调节依赖于生物对体内氮素状况信号及体外（生长介质中）氮素信号的感受。过去几十年中,在细菌、真菌中已经对氮素营养信号有了较好的研究,而植物中则相对缓慢,但也有了一定的认识。本文重点比较细菌、真菌及植物中氮信号系统的组成,以期为进一步认识植物中的氮信号接受与转导系统提供启示。     

Aircraft operations near concentrations of birds in Antarctica: The development of practical guidelines

Aircraft operations have the potential to disturb and to impact negatively on bird life. A gradient of increasing behavioural response is evident in birds when exposed to increasing aircraft stimulus. The most major disturbance is likely to lead to impacts on the health, breeding performance and survival of individual birds, and perhaps bird colonies. A process of revision to policies on aircraft operations contained in management plans for a number of specially protected areas in Antarctica by the United Kingdom, accompanied by consultations made within the scientific community through the Scientific Committee on Antarctic Research (SCAR) and with operational interests through the Council of Managers of National Antarctic Programmes (COMNAP) resulted in new guidelines being adopted by the Antarctic Treaty Consultative Parties in June 2004. The principal recommendations of the guidelines are that bird colonies should not be overflown below 2000 ft (∼610 m) above ground level and landings within 1/2 nautical mile (∼930 m) of bird colonies should be avoided wherever possible. These guidelines are less stringent and less specific than those that were recommended by the SCAR specialist group on birds, and represent a compromise to accommodate operational needs. While the adoption of clear and consistent guidelines for the operation of aircraft in Antarctica is welcome in that this provides practical advice that is likely to reduce incidences of close aircraft/bird encounters, there remains insufficient knowledge of the interactions between aircraft and birds in Antarctica, and the consequent impacts on individual birds and on bird populations. It is important, therefore, that the guidelines adopted are considered interim, and should be kept under scrutiny with revisions made as new and improved research results appear.     

The abundance of heterocystous blue-green algae in rice soils and inocula used for application in rice fields

Summary Algal populations were quantified (as colony-forming units [CFU] per square centimetre) in 102 samples of rice soils from the Philippines, India, Malaysia and Portugal, and in 22 samples of soil-based inocula from four countries. Heterocystous blue-green algae (BGA) were present in all samples. Nostoc was the dominant genus in most samples, followed by Anabaena and Calothrix. In soils, heterocystous BGA occurred at densities ranging from 1.0 × 10² to 8.0 × 10⁶ CFU/cm² (median 6.4 × 10⁴) and comprised, on average, 9% of the total CFU of algae. Their abundance was positively correlated with the pH and the available P content of the soils. In soil-based inocula, heterocystous BGA occurred at densities ranging from 4.6 × 10⁴ to 2.8 × 10⁷ CFU/g dw (dry weight), comprising only a moderate fraction (average 13%) of the total algae. In most soils, the density of indigenous N₂-fixing BGA was usually higher than that attained by applying recommended rates of soil-based inoculum. Whereas research on the practical utilization of BGA has been mostly directed towards inoculation with foreign strains, our results suggest that attention should also be given to agricultural practices that enhance the growth of indigenous strains already adapted to local environmental conditions.Maître de Recherches ORSTOM (France), Visiting Scientist at IRRI