龙眼GPX基因的克隆、原核表达及其在体胚发生过程中的表达分析

采用RT-PCR 与RACE相结合的方法,从龙眼胚性愈伤组织中克隆了长947 bp含有完整开放阅读框的龙眼DlGPX cDNA序列（GenBank登录号为EU364813）和长度为1 736 bp的DNA序列（GenBank登录号为EU680970）。其编码1个含有168个氨基酸的蛋白质。DlGPX基因中含有5个内含子,均符合真核生物内含子通用的GT-AG 法则。生物信息学分析显示：该蛋白为亲水的酸性蛋白质,定位于叶绿体;与其他植物的GPX有较高的同源性。将该基因构建成原核表达载体,经IPTG诱导表达了１个分子量约为23 ku的蛋白。利用实时荧光定量PCR（qPCR）技术研究该基因在龙眼体胚发生发育过程中的表达情况,结果显示,从松散型胚性愈伤组织到不完全胚性紧实球形结构阶段,DlGPX mRNA的转录水平逐渐升高,在胚性紧实球形结构阶段降到最低水平,子叶形胚阶段又上升到与不完全胚性紧实球形结构阶段相当的水平。     

龙眼14-3-3基因及其启动子的克隆以及在体胚发生过程中的表达分析

分离克隆龙眼(Dimocarpus longan Lour.)胚性愈伤组织14-3-3基因,并分析该基因在龙眼体胚发生过程中的表达情况。采用RT-PCR结合RACE法,获得龙眼胚性愈伤组织14-3-3基因的cDNA全长序列和DNA序列,采用TAIL-PCR法,获得龙眼胚性愈伤组织14-3-3基因的启动子DNA序列,运用生物信息学方法对序列进行分析,并通过实时荧光定量PCR(qRT-PCR)研究该基因在龙眼体胚发生过程中的表达。结果表明:经克隆得到龙眼胚性愈伤组织14-3-3基因786 bp的cDNA全长序列(GenBank检索号为GU573765),该cDNA开放阅读框推定的氨基酸序列(含261个氨基酸)与其它植物14-3-3具有较高同源性;该基因的DNA序列(GenBank检索号为GU573766)长为1 859 bp,包含3个内含子,内含子的剪切位点均符合真核生物"GT-AG"规则;该基因的启动子DNA序列(GenBank检索号为GU573766)长为910 bp;该基因在龙眼体胚各阶段均有表达,其中在松散型胚性愈伤组织阶段和心形胚及鱼雷形胚阶段呈现高表达,整个变化趋势呈"倒S"状。     

小麦UROS基因的可变剪接

可变剪接是一种重要的基因表达调控机制,对植物的发育、生理、代谢、信号转导以及外界逆境的响应等多个过程都有调控。为进一步探讨小麦基因的可变剪接机制以及深入研究小麦UROS基因的功能,以返白系、矮变1号和中国春三个普通六倍体小麦品种为材料,克隆了尿卟啉原Ⅲ合成酶基因(UROS)的gDNA和cDNA序列,并利用生物信息学技术研究了小麦叶片中UROS基因的可变剪接方式。结果表明,根据在线小麦基因组数据库,将克隆到的UROS基因组DNA序列分别定位在4BL和4DL两个基因位点,命名为UROS-4B和UROS-4D。从三个品种中克隆得到大约50个不同的UROS基因cDNA序列,通过在线预测和与基因组序列比对,发现UROS基因存在可变剪接,并鉴定出两个UROS基因位点各有一个标准剪接转录本和两个非标准剪接转录本。可变剪接方式以内含子保留为主,还有可变的供体剪接位点和可变的受体剪接位点,品种间的剪接方式不完全相同。由不同转录本预测编码的多肽氨基酸序列也存在差异,两个基因位点选取的六个转录本所编码的多肽有12个氨基酸位点的变异。     

无核荔枝凝集素基因的克隆与表达分析

应用PCR技术克隆了无核荔枝凝集素(LcLec)基因的部分cDNA(JF920723)和gDNA(JF920724)序列,序列分析表明,获得的cDNA序列含有一个468 bp的完整开放读码框结构,编码含155个氨基酸残基的多肽序列,该氨基酸残基序列与木菠萝家族的甘露糖结合凝集素同源。获得的gDNA序列含有3个内含子,长度分别为124、108和119 bp。荧光定量PCR结果表明,LcLec基因的表达量在无核荔枝果皮发育前期上升,之后下降至稳定水平;在采后果皮衰老阶段,随果皮褐变指数上升LcLec基因表达量上升。     

龙眼胚性愈伤组织ACC合成酶基因（DL-ACS1）的克隆及表达分析

以龙眼(Dimocarpus longan Lour.)胚性愈伤组织为材料,采用同源克隆和RACE方法分离得到龙眼胚性愈伤组织乙烯合成限速酶DL-ACS1(1-aminocyclopropane-1-carboxylate synthase)基因的cDNA全长序列(GenBank,FJ617537),共1 817 bp,包含了5’非编码区(73 bp),开放阅读框(1 437 bp),3’非编码区(307 bp)。该cDNA开放阅读框的氨基酸序列(含478个氨基酸)与其它植物ACS具有77%～63%同源性,属于ACC Synthase家族。实时荧光定量PCR分析表明,该基因在龙眼体细胞胚胎(以下简称龙眼体胚)各阶段的表达量存在较大的时期差异,其中,球形胚(Stage 5)的表达量最高,鱼雷形胚(Stage 7)的表达量最低。     

大豆天冬氨酸途径关键酶基因GmASADH的克隆与分析

本研究基于电子克隆获得的大豆GmASADH 基因cDNA序列,根据其编码区设计特异引物,以大豆品种小鹦哥豆总RNA为模板,通过RT-PCR 获得了约1130bp的cDNA片段,T/A 克隆后进行序列测定。测序结果显示,GmASADH基因家族有两个成员,命名为GmASADH1（FJ581425）和GmASADH2（HM015631）。GmASADH1编码区长度为1134bp,编码378个氨基酸,由7个外显子和6个内含子构成,定位于Gm08染色体。GmASADH2编码区长度为1131bp,编码377个氨基酸,由7个外显子和6个内含子构成,定位于Gm05染色体。两个基因在氨基酸水平上的相似性达到了96.02%,在二级及三级结构上均有不同程度的差异。ASADH蛋白含有NADB_Rossmann superfamily和Semialdhyde_dhC superfamily结构域。     

茶树CsASR基因的克隆及其表达分析

逆境胁迫影响茶树生长发育及茶叶品质,ASR(abscisic acid,stress,ripening-induced)基因在植物抗逆响应中具有重要功能。本研究以龙井43品种茶树为材料,从中克隆了CsASR基因的全长cDNA序列、基因组序列及其启动子序列,分析了该基因的生物信息学特征及在组织间和不同胁迫处理下的表达模式。结果显示,CsASR的cDNA序列全长875 bp,含有546 bp的ORF序列,编码181个氨基酸,蛋白质分子量19.89 kD,理论等电点5.69;CsASR蛋白结构序列中74.5%的序列为无序结构,是一种无序蛋白;CsASR的C-端含有ABA/WDS功能结构域,主要定位于细胞质和细胞核中;茶树CsASR与海枣的ASR相似性最高,为87%,而在进化树中与枣的关系最近。CsASR基因含2个外显子,第1个外显子长363 bp,第2个外显子长183 bp,内含子较大为2 750 bp,内含子中含7种简单重复序列和2种DNA转座子序列。克隆获得起始密码子ATG上游2 554 bp的启动子区序列,该启动子上含有干旱、低温、高温以及ABA等相关的顺式作用元件。CsASR在根中的表达量最低;ABA抑制CsASR的表达,而干旱、NaCl和低温胁迫能够显著上调CsASR的表达。表明CsASR基因可能与茶树抗逆密切相关。     

荔枝快速碱化因子基因的克隆与表达分析

从已构建的荔枝果皮cDNA文库中挑取克隆测序,获得一条荔枝快速碱化因子(LcRALF)基因(GenBank登录号:EU024484).该基因的cDNA全长为666 bp,含1个381 bp的开放读码结构,编码126个氨基酸.生物信息学分析表明,该基因的氨基酸序列与拟南芥、烟草等物种的RALF基因一致性较高,具有RALF基因氨基酸序列的典型特征.RT-PCR分析表明,该基因在荔枝果皮中特异表达,且在新采收的荔枝果皮中表达量最高.随着果皮的衰老该基因表达量逐渐下降.     

水稻谷蛋白基因GluB-6的cDNA克隆及表达

根据已克隆谷蛋白基因的保守氨基酸序列搜索基因组数据库,获得与之高度同源的水稻基因组序列,通过生物软件进行基因预测和验证,用RT PCR法克隆得到1个新的谷蛋白基因的cDNA克隆。核酸序列分析和体外表达结果表明,该基因cDNA序列全长为1517 bp,含有1个编码495个氨基酸残基的开放阅读框（ORF）,推导的氨基酸序列与谷蛋白基因家族的相似性介于53.6%~82.8%,并与B亚族谷蛋白基因的同源性更高,因此命名为GluB 6（GenBank注册号AY429651）。Northern杂交显示,GluB 6基因具有高度的胚乳表达特性。     

橡胶树体细胞胚胎发生相关类受体蛋白激酶基因（HbSERK1）的克隆及表达分析

从橡胶树体细胞胚中克隆了一个与体细胞胚胎发生相关的类受体蛋白激酶基因,命名为HbSERK1。HbSERK1长2 159 bp,含有一个1 482 bp的阅读框,编码493个氨基酸。HbSERK1序列与毛果杨、葡萄、胡萝卜、马铃薯、拟南芥和燕麦的体细胞胚胎发生相关类受体蛋白激酶同源性分别为91%、90%、88%、88%、87%、86%。生物信息学分析表明,HbSERK1含有一个高度保守的蛋白激酶家族结构域,同时含有ATP结合区、蛋白激酶活化位点和Ser/Thr活性位点。RT-PCR半定量分析表明,HbSERK1在叶、胶乳、雄花、生长在愈伤诱导培养基上30 d和50 d的愈伤组织及生长在胚状体诱导培养基上12 d的胚性愈伤组织中均未表达,而生长在胚状体诱导培养基上21 d和40 d的胚性愈伤组织及成熟体细胞胚中表达。橡胶树HbSERK1基因主要在体细胞胚发生的早期表达,表明其可能参与橡胶树的体细胞胚早期发生过程。     

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.     

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.     

Mineral composition of some traditional Mexican teas

Teas of plant origin traditionally consumed by the Mountain Pima of Chihuahua, Mexico, were analyzed for mineral nutritional content. Fe, Cu, Zn, Ca, and Mg composition was determined for native teas made from shoots ofTagetes lucida, T. filifolia, Elytraria imbricata, andHolodiscus dumosus, and from root xylem ofCeanothus depressus andPhaseolus ritensis. Native uses of these teas are also described.     

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.     

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.     

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.