拟南芥防御素基因PDF1.2启动子与GUS重组载体的构建与转化

PDF1.2基因在植物的防御系统中扮演着重要的角色,其编码产生的植物防御素参与了植物对真菌入侵、病毒感染、不良环境等逆境胁迫的防御反应。试验利用从拟南芥中通过PCR扩增和克隆的该基因启动子构建GUS报告基因的表达载体,通过根癌农杆菌浸渍转化法将表达报告基因转入拟南芥中,筛选获得了转化植株。获得以下主要结果:(1)成功克隆了拟南芥PDF1.2基因的启动子并将其与带GUS标记基因的载体进行了融合,得到了重组的融合载体;(2)成功的将带GUS标记基因的拟南芥PDF1.2基因启动子融合载体转入到拟南芥植株中并利用其自带抗性标记筛选到了抗性植株;(3)利用GUS染色技术检测了转基因植株中PDF1.2基因启动子的表达情况。     

大豆组织特异启动子的克隆与功能分析

采用同源序列法克隆得到了大豆根部特异性启动子、种皮特异性启动子、种子特异性启动子,核苷酸序列大小分别为2 500bp、1 832bp、1 268bp,分别具有CANNTG-motifs、GATA-box、ACGT等启动子表达元件,并分别构建了这3个组织特异性启动子的植物报告表达载体。通过农杆菌介导法将3个表达载体转入烟草NC89,通过组织化学染色和GUS基因的相对表达量分析验证3个启动子的功能。通过转化获得了根部特异性启动子转基因烟草植株11株,种皮特异性启动子转基因烟草植株4株,种子特异性启动子转基因烟草植株8株,转35S启动子启动GUS基因的阳性烟草植株7株。GUS化学组织染色结果表明,克隆得到的根、种皮、种子特异性启动子都表现为组织特异性表达,表达水平明显高于叶片、茎部、花等部位。进一步的荧光定量PCR结果显示,根部特异性启动子GUS的相对表达量在根部最高,而在茎、叶、种子、种皮、花中表达量较低;种皮部特异性启动子GUS的相对表达量在种皮最高,而在根、茎、叶、种子、花中表达量较低;种子特异性启动子GUS的相对表达量在种子最高,而在根、茎、叶、种皮、花中表达量较低,但是与35S启动子相比,这3个特异性启动子的GUS相对表达量在相应的组织均低于35S启动子。     

甘蓝型油菜胚胎特异启动子pBnaA09g21960D的分析

为筛选甘蓝型油菜胚胎特异性表达启动子,以甘蓝型油菜冬性品种Darmor的叶片基因组为模板,克隆了Bna A09g21960D基因上游990bp的启动子序列。顺式调控元件的预测分析表明,该启动子除具有核心调控元件TATA box和CAAT-box外,还含多个组织特异性、激素、逆境、光合作用等表达相关的顺式作用元件,如ABRE、GCN4_motif、TGACG-motif等。为了验证该基因启动子的表达模式,构建了由其驱动GUS报告基因的植物融合表达载体DX2181-p Bna A09g21960D,并采用农杆菌介导的花序浸染法转化野生型拟南芥。对筛选和鉴定的阳性转基因株系进行GUS组织化学法染色,结果表明GUS活性在拟南芥胚胎中特异表达,而在其他组织和器官均未表达,这表明p Bna A09g21960D具有驱动GUS报告基因在拟南芥种子胚胎中特异表达的潜质。这为该启动子在转基因技术提高油菜品质方面和人工创建种质资源等方面的应用提供了较好的基础。     

玉米醇溶蛋白ze19基因启动子克隆与功能分析

从玉米自交系9801基因组DNA中克隆玉米醇溶蛋白基因ze19启动子,将其克隆到pMD18-T载体上.该序列与发表序列同源性为94％,包括TATA box、CAAT box启动子基本元件以及多种参与调节醇溶蛋白表达的顺式元件.将ze19启动子取代质粒PBI 121-gus中的CaMV35S启动子,构建由ze19启动子驱动GUS的植物表达载体pBIze19-gus,利用土壤杆菌介导法将重组载体pBIze19-gus转入烟草中.对转基因烟草植株GUS活性的定性与定量分析结果表明,ze19启动子驱动GUS基因在转基因烟草种子中表达活性最高,在叶片中活性很弱,在茎中没有活性.所克隆的ze19启动子具有种子特异表达特性,为玉米种子生物反应器的研究提供借鉴.     

拟南芥HGO基因启动子与GUS融合表达载体的构建及转化鉴定

HGO基因编码的尿黑酸1,2双加氧酶催化酪氨酸降解途径中的倒数第三步。本研究构建了拟南芥HGO基因启动子与GUS的融合表达载体,采用农杆菌介导法转化拟南芥获得转基因株系。经GUS组织化学染色鉴定,证明拟南芥HGO基因启动子与GUS的融合表达载体成功构建且能正常启动GUS基因的表达,为深入研究拟南芥HGO基因的表达特征创建了有价值的材料。     

拟南芥CWINV1启动子GUS表达载体构建及转基因植株的鉴定

CWINV是编码细胞壁蔗糖转化酶的基因。为深入探讨CWINV基因的表达调控机理,构建了拟南芥CWINV1基因启动子的GUS融合表达载体并转入拟南芥,获得了转基因植株。GUS染色的结果进一步验证了重组表达载体的正确性和实用性。     

拟南芥AtLURP1基因启动子在转基因烟草和水稻中的功能分析

克隆拟南芥LURP1基因上游1515 bp的启动子调控序列并命名为AtLURP1p,将其与GUS报告基因融合,构建成植物表达载体,分别遗传转化烟草和水稻,获得LURP1p::GUS的烟草和水稻转基因植株及其相应的T2代株系,分别研究LURP1p对水稻稻瘟病、辣椒青枯病侵染及SA、MeJA、ABA等几种重要的植物激素信号分子处理的应答.结果表明:(1)转基因烟草和水稻在几种激素,包括SA、MeJA、ABA的诱导处理下,GUS基因均可以被诱导表达;(2)转基因烟草在细菌性病菌青枯病的侵染下,GUS可被诱导表达并表现出持续表达的趋势,转基因水稻在稻瘟病的侵染下,其GUS基因也被诱导表达,并表现出后期持续上调的趋势.这些研究结果表明,拟南芥LURP1的应答逆境信号通路也存在于烟草和水稻等植物,该启动子可用作诱导型启动子广泛地应用于不同植物抗病基因工程.     

大豆GmGBP1启动子受激素和外源环境诱导的研究

采用PCR方法克隆大豆光周期GmGBP1基因启动子,构建含有GmGBP1启动子驱动报告基因GUS的融合性表达载体pBI121-pGmGBP1::GUS,通过花序浸泡法,转化野生型拟南芥获得pBI121-pGmGBP1::GUS转基因植株,对T3代转基因拟南芥进行GUS染色,研究了GmGBP1启动子受激素和外源环境诱导的表达规律。结果表明:大豆GmGBP1启动子受水杨酸(SA)和干旱(PEG)以及高温等外源环境的诱导,与生物信息学预测GmGBP1启动子序列存在的逆境胁迫响应元件的结果相一致。     

拟南芥AtNUDT8启动子的分离及其功能分析

根据已发表的拟南芥基因组序列设计合成一对引物,以拟南芥基因组DNA为模板克隆AtNUDT8上游非编码区,并与pBAR-GUS3相连构建了植物表达载体pNUDT8P-GUS,采用农杆菌GV3101介导的渗透法转化野生型拟南芥,通过除草剂筛选获得了一批抗性纯合子转基因植株,再对转基因植株2周幼苗进行GUS活性的组织化学染色分析,并对3.5~4周的转基因植株叶片进行病原菌诱导表达分析。结果表明：已获得AtNUDT8启动子,该启动子为组成型表达启动子,并且病原菌Pst.DC3000对该启动子无诱导作用。     

启动子陷阱技术在橡胶树中的应用研究

启动子陷阱技术是克隆未知基因启动子的有效途径。首先用反向无启动子的GUSA替换pCAMBIA-2301载体上的GUSA及启动子序列，构建橡胶树启动子陷阱载体pCAMBIA2301：GUS-NOS，然后转化橡胶树。结果表明：获得了GUS染色阳性胚状体6个，再生后获得2个转基因株系。其中1个株系在胚状体、叶片和根中能检测到GUS基因的强烈表达，另1个株系仅在根中检测到微弱的GUS基因表达，说明获得的转基因植株为不同启动子启动GUS基因的株系。因此通过橡胶树启动子陷阱载体的构建及其在橡胶树中的表达，能够发现和克隆橡胶树内源基因的启动子。     

Comparative Assessment of the Efficacy of Various Nitrogen Fertilizers

Summary

The efficiency of N fertilizers is usually poor; often less than 50% of the applied N is taken up by the crop. This review focuses on various N fertilizers with respect to the significance of different N loss pathways, namely (i) ammonia volatilization, (ii) dinitrogen and nitrogen oxide emissions, and (iii) nitrate leaching. Further, the significance of biological N immobilization, ammonium fixation and, finally, the impact of nitrate vs. ammonium uptake on crop yield are also discussed. The reviewed literature shows that N fertilizers may differ markedly in their susceptibility to losses. There is, however, considerable scope to improve N efficiency of each N source by proper N management practices.     

Studies on natural repellents against potato tuber moth (Phthorimaea operculella Zeller) in country stores

Summary The effects of the leaves of five plant species, one from each of the generaAmbrosia, Anemone, Eupatorium, Eucalyptus andLantana, on potato tuber moth were investigated under indigenous storage conditions at the Central Potato Research Station, Shillong (1800 m above sea level). Their action was compared with that of a biological insecticide (spores ofBacillus thuringiensis), a chemical insecticide (carbaryl), and an untreated control. The data collected after six months storage on tuber damage, sprout damage and the rotting indicated that the leaves ofLantana aculeata provided most protection to the tubers, reducing damage from over 70% in the check to below 5%, and sprout damage from over 45% to below 3%. Next best wasEucalyptus globulus followed byB. thuringiensis. They may be used on tubers stored for table use or for seed as they had no adverse effect on germination or on the yield of a subsequent crop.     

Novel food and non-food uses for sorghum and millets

Sorghum and millets have considerable potential in foods and beverages. As they are gluten-free they are suitable for coeliacs. Sorghum is also a potentially important source of nutraceuticals such antioxidant phenolics and cholesterol-lowering waxes. Cakes, cookies, pasta, a parboiled rice-like product and snack foods have been successfully produced from sorghum and, in some cases, millets. Wheat-free sorghum or millet bread remains the main challenge. Additives such as native and pre-gelatinised starches, hydrocolloids, fat, egg and rye pentosans improve bread quality. However, specific volumes are lower than those for wheat bread or gluten-free breads based on pure starches, and in many cases, breads tend to stale faster. Lager and stout beers with sorghum are brewed commercially. Sorghum's high-starch gelatinisation temperature and low beta-amylase activity remain problems with regard to complete substitution of barley malt with sorghum malt . The role of the sorghum endosperm matrix protein and cell wall components in limiting extract is a research focus. Brewing with millets is still at an experimental stage. Sorghum could be important for bioethanol and other bio-industrial products. Bioethanol research has focused on improving the economics of the process through cultivar selection, method development for low-quality grain and pre-processing to recover valuable by-products. Potential by-products such as the kafirin prolamin proteins and the pericarp wax have potential as bioplastic films and coatings for foods, primarily due to their hydrophobicity.     

In vitro antimicrobial activity of different accessions ofSolanum commersonii Dun. from Uruguay

Summary The in vitro antimicrobial activity of extracts from accessions ofSolanum commersonii Dun. collected in the south of Uruguay was investigated against five microorganisms including the pathogenRalstonia solanacearum. A total of 30 extracts corresponding to organic and aqueous extracts were studied. Interestingly, most of the positive results for growth inhibition were againstR. solanacearum. The extracts were also analyzed for the presence of glycoalkaloids and lectins. Six of the ten aqueous extracts showed lectin presence and a wide variation in the type and amounts of glycoalkaloids, was found. Results indicate that there is no clear relationship between the antimicrobial activity against the five microorganisms screened and the presence or amounts of lectins and glycoalkaloids, traditionally regarded as possible antimicrobial metabolites in theSolanum genus, which indicates the presence of as yet unidentified antimicrobial compounds.     

Extreme resistance to infection by potato virus X in genotypes of wild tuber-bearingSolanum species

Summary Clones derived from thirty-one different accessions (nineteen of Argentine origin) belonging to eightSolanum species were screened for resistance to infection by potato virus X strain cp (PVX cp) by mechanical inoculation of plantlets that had been micropropagated in vitro. Estimates of PVX multiplication obtained by enzyme linked immunosorbent assay and slot blot nucleic acid hybridization allowed the identification of resistant clones derived from five accessions belonging toS. commersonii S. oplocense, S. sparsipilum andS. tuberosum andigena. Resistant genotypes supported PVX concentrations 5 to 15 times smaller than did the susceptible control cultivar Spunta. Graft inoculation test confirmed the presence of extreme resistance similar to that conferred by the ‘immunity’ gene X¹ (also called RX_act).     

Cyanobacteria Secondary Metabolites as Biotechnological Ingredients in Natural Anti-Aging Cosmetics: Potential to Overcome Hyperpigmentation,Loss of Skin Density and UV Radiation-Deleterious Effects

The loss of density and elasticity, the appearance of wrinkles and hyperpigmentation are among the first noticeable signs of skin aging. Beyond UV radiation and oxidative stress, matrix metalloproteinases (MMPs) assume a preponderant role in the process, since their deregulation results in the degradation of most extracellular matrix components. In this survey, four cyanobacteria strains were explored for their capacity to produce secondary metabolites with biotechnological potential for use in anti-aging formulations. Leptolyngbya boryana LEGE 15486 and Cephalothrix lacustris LEGE 15493 from freshwater ecosystems, and Leptolyngbya cf. ectocarpi LEGE 11479 and Nodosilinea nodulosa LEGE 06104 from marine habitats were sequentially extracted with acetone and water, and extracts were analyzed for their toxicity in cell lines with key roles in the skin context (HaCAT, 3T3L1, and hCMEC). The non-toxic extracts were chemically characterized in terms of proteins, carotenoids, phenols, and chlorophyll a, and their anti-aging potential was explored through their ability to scavenge the physiological free radical superoxide anion radical (O₂^•−), to reduce the activity of the MMPs elastase and hyaluronidase, to inhibit tyrosinase and thus avoid melanin production, and to block UV-B radiation (sun protection factor, SPF). Leptolyngbya species stood out for anti-aging purposes: L. boryana LEGE 15486 presented a remarkable SPF of 19 (at 200 µg/mL), being among the best species regarding O₂^•− scavenging, (IC₅₀ = 99.50 µg/mL) and also being able to inhibit tyrosinase (IC₂₅ = 784 µg/mL), proving to be promising against UV-induced skin-aging; L. ectocarpi LEGE 11479 was more efficient in inhibiting MMPs (hyaluronidase, IC₅₀ = 863 µg/mL; elastase, IC₅₀ = 391 µg/mL), thus being the choice to retard dermal density loss. Principal component analysis (PCA) of the data allowed the grouping of extracts into three groups, according to their chemical composition; the correlation of carotenoids and chlorophyll a with MMPs activity (p < 0.01), O₂^•− scavenging with phenolic compounds (p < 0.01), and phycocyanin and allophycocyanin with SPF, pointing to these compounds in particular as responsible for UV-B blockage. This original survey explores, for the first time, the biotechnological potential of these cyanobacteria strains in the field of skin aging, demonstrating the promising, innovative, and multifactorial nature of these microorganisms.     

Marine Pharmacology in 2009–2011: Marine Compounds with Antibacterial,Antidiabetic, Antifungal,Anti-Inflammatory,Antiprotozoal, Antituberculosis,and Antiviral Activities; Affecting the Immune and Nervous Systems,and other Miscellaneous Mechanisms of Action ?

The peer-reviewed marine pharmacology literature from 2009 to 2011 is presented in this review, following the format used in the 1998–2008 reviews of this series. The pharmacology of structurally-characterized compounds isolated from marine animals, algae, fungi and bacteria is discussed in a comprehensive manner. Antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral pharmacological activities were reported for 102 marine natural products. Additionally, 60 marine compounds were observed to affect the immune and nervous system as well as possess antidiabetic and anti-inflammatory effects. Finally, 68 marine metabolites were shown to interact with a variety of receptors and molecular targets, and thus will probably contribute to multiple pharmacological classes upon further mechanism of action studies. Marine pharmacology during 2009–2011 remained a global enterprise, with researchers from 35 countries, and the United States, contributing to the preclinical pharmacology of 262 marine compounds which are part of the preclinical pharmaceutical pipeline. Continued pharmacological research with marine natural products will contribute to enhance the marine pharmaceutical clinical pipeline, which in 2013 consisted of 17 marine natural products, analogs or derivatives targeting a limited number of disease categories.     

Recent Modifications of Chitosan for Adsorption Applications: A Critical and Systematic Review

Chitosan is considered to be one of the most promising and applicable materials in adsorption applications. The existence of amino and hydroxyl groups in its molecules contributes to many possible adsorption interactions between chitosan and pollutants (dyes, metals, ions, phenols, pharmaceuticals/drugs, pesticides, herbicides, etc.). These functional groups can help in establishing positions for modification. Based on the learning from previously published works in literature, researchers have achieved a modification of chitosan with a number of different functional groups. This work summarizes the published works of the last three years (2012–2014) regarding the modification reactions of chitosans (grafting, cross-linking, etc.) and their application to adsorption of different environmental pollutants (in liquid-phase).