Polymorphisms in the ALS gene of weedy rice (Oryza sativa L.) accessions with differential tolerance to imazethapyr

Herbicide-resistant Clearfield™ rice technology allows the use of ALS inhibitors to control weedy rice. Weedy rice plants have differential tolerance to imazethapyr, perhaps due to ALS polymorphisms. We aimed to assess ALS polymorphisms in weedy rice accessions from Arkansas, USA, with differential tolerance to imazethapyr in seedling growth bioassays. Six base changes were identified in the ALS of 14 weedy rice accessions. Three of these nucleotide changes resulted in amino acid substitutions — Pro⁹³Thr, Glu⁶³⁰Asp, and Val⁶⁶⁹Met — in four accessions: Ark-4, Ark-9, Poi-1 and Poi-6. The GR₅₀ values and inhibition of root and shoot growth (%) of these accessions differed. The Glu⁶³⁰Asp substitution occurs in the herbicide binding domain B and Val⁶⁶⁹Met occurs at the C-terminal tail where the co-factor binds. Variability in weedy rice ALS exists, but polymorphism patterns did not relate to tolerance levels. The observed mutations presented the possibility that sustained selection pressure will eventually lead to selection of a herbicide-tolerant individual that will be the progenitor of a resistant population. Concomitantly, pollen-mediated gene flow from Clearfield™ rice to weedy rice will lead to the evolution of ALS-resistant weedy rice populations.     

Molecular characterization and comparative analysis of four new genes from Sec2 locus encoding 75K γ-secalins of rye species

Using a PCR-based strategy, four new 75K γ-secalin genes were isolated from Secale cereale, Secale vavilovii, Secale sylvestre, and Secale strictum in genus Secale (rye). Based on amino acid sequences, the primary structure of the 75K γ-secalin subunits was demonstrated, which was composed of four main structural regions: (a) a conservative 19 amino acids signal peptide, (b) a steady short N-terminal region of 12 amino acids containing a cysteine residue, (c) a repetitive domain, which began with the conservative tetrapeptides PQ₃ and was rich in glutamine and proline. PFPQ₁₋₂(PQQ)₁₋₂ was the core repeat motif in the repetitive region. Besides amino acid substitutions, this region showed variations in length due to the insertion and deletion events. In the repetitive region of EF432549 (Secale strictum), there were two octapeptides (PFPQQPQQ and PVPQQSQQ) insertions. On the contrary, deletion events of two residues (QT) took place in EF432546 (Secale sylvestre). Accounting for the amino acid replacement, an extra cysteine residue appeared in the repetitive region of EF432546, which did not exist in other genes, and (d) a conserved 143 amino acids C-terminal domain including eight cysteine residues. The implications of the results for quality improvement are discussed.     

磷对NaCl胁迫下玉米幼苗渗透调节能力的影响

研究不同供磷水平对NaCl胁迫下玉米幼苗有机渗透调节物质和离子含量的影响。结果表明,盐胁迫下低磷处理玉米幼苗叶片中可溶性糖和游离氨基酸增加,根系中显著降低;增加供磷水平,叶片中可溶性糖和游离氨基酸含量下降,根系中含量上升,同时叶片和根系中可溶性蛋白含量增加。磷可降低盐胁迫下玉米幼苗各器官中的Na~+含量,同时增加各器官的K~+、Ca~(2+)和Mg~(2+)含量,降低Na~+/K~+与Na~+/Ca~(2+)比值。磷有助于维持植株的碳氮代谢平衡,促进有机渗透调节物质的运输与分配,改善各器官的离子平衡,增强植株的渗透调节能力,从而缓解盐胁迫带来的伤害。     

Characterisation of a ω-gliadin gene in Triticum tauschii

A ω-gliadin gene at the Gli-D^t1 locus of Triticum tauschii accession AUS18913 was isolated using PCR primers, designed from published sequences of ω-gliadin genes of bread wheat cv Cheyenne, and deduced sequences of the N-terminal amino acids of ω-gliadin proteins. Further, the derived protein was isolated from A-PAGE and was sequenced. The protein sequence contained a signal peptide of 19 amino acids followed by a short N-terminal sequence of 11 amino acids, a central repetitive domain that covers approximately 90% of the sequence and a short C-terminal domain of 12 amino acids. The sequence comparison with other ω-gliadins showed a high level of similarities between them. Further analysis of the ω-gliadins using A-PAGE revealed that there are three ω-gliadin proteins in AUS18913 accession. Comparison of N-terminal sequences of these proteins revealed that two of these proteins have very high homologies with ω-gliadins of Cheyenne while the third one was significantly different.     

大麦转录因子基因 HvNAC1的克隆及功能初探

NAC转录因子家族是植物中最大的转录因子家族之一,在植物的生长发育及植物参与生物与非生物胁迫过程中起重要作用。本研究通过分析感染大麦温和花叶病毒(Barley mild mosaic virus,BaMMV)的大麦转录组测序结果,获得表达上调的基因 HORVU5Hr1G011650,基因注释为 HvNAC1。通过生物信息学分析发现该基因全长915 bp,编码304个氨基酸,分子量为33.3 kDa,理论等电点为9.21,在14～141位氨基酸之间含有NAC转录因子家族保守结构域。系统进化分析发现,该基因与小麦、拟南芥中的NAC转录因子同源性较高。组织表达分析发现,该基因在大麦的不同生长时期均有表达,在结实后期和外颖壳中表达量较高。农杆菌介导的烟草亚细胞定位实验表明,该基因定位于细胞核中。在酵母实验中,发现 HvNAC1具有完整的转录因子活性。     

Studies on samh seeds (Mesembryanthemum forsskalei Hochst) growing in Saudi Arabia: 2: Chemical composition and microflora of samh seeds

The chemical composition of samh seed have been investigated. Proximate analysis showed a composition of 22.25% protein, 5.7% moisture, 5.6% fat, 4.0% ash, 9.7% crude fiber, and the remainder being total carbohydrates. Mineral element analysis revealed that potassium, magnesium, sodium and calcium were present as the major elements. Iron, manganese, zinc and copper were found at lower levels. However, lead was not detected in the samh seeds. Gas-liquid chromatographic analysis of the methylester of the fatty acids of the samh seeds oil revealed the presence of fourteen fatty acids. Linoleic and oleic acids were the principle unsaturated fatty acids. While palmitic acid was the main saturated fatty acid. Amino acid analysis of the samh seeds showed the presence of seventeen amino acids including eight essential amino acids. Glutamic acid, arginine, and aspartic acid were the major amino acids. Cystine and proline were present in trace amounts. These results some of which have not been reported elsewhere indicate the high nutritional potential of Saudi samh seeds. The total aerobic bacterial count and total sporeformers of seeds were 19×10⁷ and 5×10⁴ cfu/g respectively, thus the enterobacteriaceae,B cereus and yeast and molds were 5×10², 1×10² and 7×10² respectively. The seeds were Staph. free and the samh extract had no antimicrobial effect.     

普通小麦TaADF8基因的克隆及表达分析

肌动蛋白解聚合因子(actin-depolymerizing factor,ADF)普遍存在于真核细胞中,为低分子量的肌动蛋白结合蛋白,在调控细胞内肌动蛋白纤维的聚合和解聚中起关键作用。为给深入研究TaADF8基因在小麦中的功能机理奠定基础,并为进一步丰富小麦ADF基因研究内容提供理论参考,本研究利用电子克隆策略从小麦品种CP53中克隆出TaADF8基因(GenBank登录号为KJ864962)后对其进行序列分析,并进一步采用荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术对其在小麦不同组织间的表达差异及不同非生物胁迫下的表达模式进行分析。核酸序列分析表明,该基因全长695bp,拥有完整的ORF,编码142个氨基酸。氨基酸序列分析表明,该蛋白含有保守的ADF同源区和PIP2结合结构域,且在氨基端有核定位信号。进化和聚类分析表明,小麦TaADF8基因与大麦HvADF2基因、HvADF3基因和水稻OsADF3基因亲缘关系较近,蛋白相似度分别为75.35%、93.66%和67.86%。qRT-PCR表达特性分析显示,该基因为组成型表达,在根、茎、叶、颖壳和雄蕊中均表达,且在根、叶和雄蕊中表达量较高;该基因表达受低温的强烈诱导,同时也受水分、高盐和外源脱落酸胁迫诱导。     

Protein Digestibility using Corrected Amino Acid Score method (PDCAAS) of four types of mushrooms grown in Jordan

This study was conducted to evaluate the protein quality of four types ofedible mushrooms common in Jordan in terms of protein digestibility –corrected amino acid score (PDCAAS), which is a combination of thechemical score of the limiting amino acid multiplied by true digestibility ofthe protein. True protein digestibility values were low (61.4, 73.4, 52.6and 80.5 for Terfezia claveryi, Pleurotus ostreatus, Tricholoma terrum and Agaricus macrosporus, respectively). Based onthe essential amino acids pattern requirements for children, the limitingamino acids in P. ostreatus and A. macrosporus protein weresulphur-containing amino acids with chemical scores of 0.61 and 0.50,respectively. However, lysine was the limiting amino acid in the protein ofT. claveryi and T. terreum with chemical scores of 0.71 and 0.67,respectively, and the PDCAAS was 0.43, 0.45, 0.35 and 0.40 for themushrooms types, respectively. Considering the pattern of essential amino acid requirements of laboratory rats, thesulphur containing amino acids were the limiting amino acids in the proteinof T. claveryi, P. ostreatus, T. terreum and A.macrosporus with chemical scores of 0.56, 0.30, 0.34 and 0.25,respectively. The PDCAAS were 0.34, 0.22, 0.17 and 0.20,respectively. It is concluded that the four mushroom types studied are of lowprotein quality.     

The effects of intravenous administration of amino acids and glucose on the milk production of dairy cows consuming diets based on grass silage

Two experiments, using intravenous infusion of nutrients, were carried out with the aim of separating milk production responses due to the provision of amino acids as precursors of milk protein synthesis from those due to the provision of amino acids as glucose precursors. Diets were based on grass silage of restricted fermentation and barley‐based supplements because it has been suggested that these diets might provide insufficient glucose precursors to meet the needs of lactose synthesis. The silages used in the experiments were of similar lactic acid contents [62 and 63 g kg^–1 dry matter (DM)] but of different water‐soluble carbohydrate (WSC) contents (206 and 20 g kg^–1 DM in Experiments 1 and 2 respectively). In Experiment 1, four dairy cows were given the following treatments in a 4 × 4 Latin square arrangement with periods of 10 d: (1) basal diet (Basal), (2) Basal plus jugular infusion of 182 g d^–1 of amino acids simulating casein (TAA), (3) Basal plus 101 g d^–1 of essential amino acids (EAA), being the essential amino acid component of the TAA treatment and (4) Basal plus 101 g d^–1 of essential amino acids plus 50 g d^–1 of glucose (EAA + G), being the glucose equivalent of the non‐essential amino acid component of treatment TAA. All infusions increased (P < 0·05) the concentration of milk protein compared with Basal but only for TAA was the increase in the yield of milk protein statistically significant (P < 0·05), amounting to 68 g d^–1. Both TAA and EAA reduced (P < 0·05) the concentration of milk fat. There was no difference between EAA and EAA + G treatments. In Experiment 2, five dairy cows were given the following treatments in a 5 × 5 Latin square design with periods of 7 d: (1) basal diet (Basal), (2) Basal plus 182 g d^–1 of amino acids simulating casein (TAA), (3) Basal plus 182 g d^–1 of non‐essential amino acids as in casein (NEAA), (4) Basal plus 100 g d^–1 of glucose (G100) and (5) basal plus 230 g d^–1 of glucose (G230). G100 supplied the glucose equivalent of NEAA whereas G230 supplied the caloric equivalent of NEAA. Again, only for TAA was the increase in yield of milk protein statistically significant (P < 0·05), amounting to 83 g d^–1. Neither glucose treatment caused any statistically significant (P > 0·05) effect on the yield of milk protein nor the yield of milk lactose. It is concluded that, in both experiments, the primary nutritional limitation on milk protein output was the supply of amino acids as precursors of milk protein, there being no evidence to support a primary limitation due to glucose supply.     

燕麦等五种谷物的氨基酸含量综合评价

为了解不同谷物氨基酸的综合质量,测定了燕麦、荞麦、小麦、大麦和水稻等5种谷物的蛋白质和氨基酸含量,并采用主成分分析、聚类分析法进行谷物氨基酸含量综合评价。结果表明,五种谷物中均含有16种氨基酸,总氨基酸含量平均为148.14 g·kg^-1,其中燕麦总氨基酸平均含量最高,为249.93 g·kg^-1;燕麦中支链氨基酸平均含量达到31.23 g·kg^-1,显著高于五种谷物支链氨基酸平均含量(21.84 g·kg^-1)。通过主成分分析,供试材料氨基酸的综合评分表现为燕麦>小麦>荞麦>大麦>水稻。经聚类分析,燕麦的氨基酸含量与其他谷物显著不同。综合来看,燕麦在蛋白质、赖氨酸、支链氨基酸、必需氨基酸、总氨基酸含量和氨基酸六个方面均优于其他4种谷物。     

小麦细胞色素P450(TaP450)基因的克隆与序列分析

细胞色素P450是一种多功能氧化酶,在植物体内担当着生物合成、代谢解毒以及抗逆等重要功能。本研究采用RACE方法从普通小麦中同源克隆到一个新的P450基因,命名为TaP450(Genbank No.KJ541960)。该基因基因组序列全长为2 643bp,含有2个外显子和1个内含子,cDNA全长为2 033bp,含有一个1 557bp的开放读码框,推导蛋白含518个氨基酸;序列分析发现其具有保守的P450结构域,但该蛋白与已报道的小麦P450蛋白序列有较大差异,表明它是小麦P450家族的新成员;蛋白结构特征分析发现,该蛋白的分子量为57.092kD,等电点为8.63,N端具有一个含29个氨基酸的跨膜结构和一个含22个氨基酸的信号肽,亚细胞定位分析表明该蛋白属于分泌蛋白。其在小麦叶片、茎、根与种子中均有表达,但在叶片和茎中表达量最高;在胁迫处理下,该基因的表达量均上调,且在盐胁迫处理下上调尤为显著,表明该基因与盐胁迫密切相关。     

玉米肉桂醇脱氢酶基因ZmCAD4的克隆和生物信息学分析

肉桂醇脱氢酶(CAD)是木质素生物合成途径的限速酶。对玉米B73基因组数据库和NCBI数据库中注释为玉米B73 CAD的基因进行挖掘和筛选，对其中1条玉米肉桂醇脱氢酶基因ZmCAD4克隆，并进行生物信息学分析。结果表明，共筛选到12条玉米CAD基因，克隆的ZmCAD4基因编码区全长1 104 bp，编码367个氨基酸。ZmCAD4蛋白含有典型的CAD1结构域、Zn结合位点和NADP结合位点，不含有跨膜结构域和信号肽，为非分泌蛋白，亚细胞定位预测定位在细胞质中。蛋白三级结构预测与拟南芥AtCAD5蛋白相似，相似度达到73.80%。系统进化分析表明，ZmCAD4与芒草、高粱等禾本科植物亲缘关系最近。     

氨基酸对小麦成熟胚愈伤组织诱导率的影响

为优化小麦成熟胚愈伤组织诱导体系,以中国春（Chinese Spring）成熟种子为材料,MS培养基为基础培养基,研究酒精、双氧水不同的灭菌时间和不同氨基酸组分对小麦成熟胚愈伤组织诱导与分化的影响。结果表明,当75%酒精浸泡时间控制在180 s、30%双氧水灭菌时间15 s以上时和当30%双氧水消毒时间控制在180 s、75%酒精浸泡时间15 s以上时,成熟胚均不会出现污染。甘氨酸、天冬氨酸、脯氨酸和亮氨酸是小麦成熟胚诱导的必须氨基酸。正交实验结果表明,培养基中最佳的氨基酸配组为甘氨酸60.0 mg·L^-1、天冬氨酸2.0 mg·L^-1、脯氨酸2.0 mg·L^-1和亮氨酸0.1 mg·L^-1。利用该培养体系,小麦成熟胚的出愈率达到97%以上。本研究优化了小麦成熟胚的培养体系,也为研究小麦氨基酸代谢的生理过程提供了参考。     

Kunitz Soybean Trypsin Inhibitor is Modified at its C-terminus by Novel Soybean Thiol Protease (Protease T1)

Abstract

Kunitz soybean trypsin inhibitor (KSTI) is hydrolyzed during seed germination to yield amino acids needed to support initial seedling growth. The type of KSTI from Glycine max (L.) Merrill cv. Toyokomachi is KSTI-Ti ^b. The KSTI-Ti ^b from 4-day-old post-germination cotyledons (KSTI-Ti ^b’) has 3 or 4 amino acid residues cleaved off at the C-terminus. This KSTI modification is important to understand the mechanism of degradation in seed reserve proteins by proteases. Protease K1 also cleaves amino acid residues at the C-terminus of KSTI but it removes 5 amino acid residues. Therefore, we presumed the KSTI-Ti ^b’ was produced by a protease other than protease K1. In this study, the protease T1 responsible for cleavage of KSTI-Ti ^b at the C-terminus was purified. The enzyme was estimated to have a molecular mass of 33 kDa from its mobility on SDS-PAGE gels. The N-terminal amino acid sequence of the purified protease T1 corresponded to amino acids Phe-73 to Phe-92 of both thiol protease isoforms A and B from the soybean leaf, and shared 83% identity with the partial amino acid sequence of the membrane-associated cysteine protease from mung bean seedlings, a protease known to perform post-translational cleavage of C-terminal peptides of target proteins. Finally, this enzyme was shown to convert KSTI-Ti ^b to KSTI-Ti ^b’.     

燕麦AsSnRK2.7编码蛋白的分子特征及基因表达特异性研究

蔗糖非发酵相关的蛋白激酶2（SnRK2）通过磷酸化在植物胁迫信号转导途径中起关键作用。为发掘并利用燕麦中的 SnRK2基因，本研究基于燕麦转录组数据的注释信息，利用RT-PCR技术从燕麦品种美达中克隆了 AsSnRK2.7基因，借助生物信息学、瞬时表达及实时荧光定量RT-PCR（qRT-PCR）技术分别对该基因或其编码蛋白进行分子特征分析、亚细胞定位和表达特异性研究。结果表明， AsSnRK2.7基因包含一个1 074 bp的开放阅读框，编码357个氨基酸，预测其编码蛋白含有一个STKc_SnRK2结构域和一个PKc_like superfamily结构域，属于SnRK2蛋白激酶家族成员。AsSnRK2.7蛋白一级序列中包含多个SnRK2家族特有的重要功能区。AsSnRK2.7蛋白与水稻的SnRK2蛋白激酶相似性最高，属于Group Ⅰ（SnRK2b）亚家族。亚细胞定位结果显示，AsSnRK2.7蛋白主要定位在细胞核中。qRT-PCR结果显示， AsSnRK2.7基因为组成型表达，在根、茎、叶和穗中的最大表达量分别出现在苗期、分蘖期、抽穗期和灌浆期；此外， AsSnRK2.7基因的表达不被ABA激活，但可以积极应答PEG、盐和低温胁迫。以上结果说明， AsSnRK2.7基因可能作为一个调节因子，通过非依赖ABA途径来调节干旱、盐和低温所引发的信号传导。     

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20–280 μm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg⁻¹ by day 13, exceeding the European Food Safety Authority recommended concentration of 44 μg kg⁻¹ in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.     

Recent Developments on the Synthesis and Bioactivity of Ilamycins/Rufomycins and Cyclomarins,Marine Cyclopeptides That Demonstrate Anti-Malaria and Anti-Tuberculosis Activity

Ilamycins/rufomycins and cyclomarins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclomarins are also very potent inhibitors of Plasmodium falciparum. Biosynthetically the cyclopeptides are obtained via a heptamodular nonribosomal peptide synthetase (NRPS) that directly incorporates some of the nonproteinogenic amino acids. A wide range of derivatives can be obtained by fermentation, while bioengineering also allows the mutasynthesis of derivatives, especially cyclomarins. Other derivatives are accessible by semisynthesis or total syntheses, reported for both natural product classes. The anti-tuberculosis (anti-TB) activity results from the binding of the peptides to the N-terminal domain (NTD) of the bacterial protease-associated unfoldase ClpC1, causing cell death by the uncontrolled proteolytic activity of this enzyme. Diadenosine triphosphate hydrolase (PfAp3Aase) was found to be the active target of the cyclomarins in Plasmodia. SAR studies with natural and synthetic derivatives on ilamycins/rufomycins and cyclomarins indicate which parts of the molecules can be simplified or otherwise modified without losing activity for either target. This review examines all aspects of the research conducted in the syntheses of these interesting cyclopeptides.     

小麦自主开花基因TaFLD单核苷酸多态性分析

为了给培育具有广泛生态适应性的小麦品种提供参考依据,通过比对拟南芥自主开花基因(FLD基因)与小麦D基因组序列,获得小麦FLD基因的同源序列.分析结果表明,小麦TaFLD基因全长8 392 bp,具有3 087 bp完整ORF,编码1 028个氨基酸,亚细胞定位预测显示,其编码蛋白定位于细胞质.TaFLD基因功能预测表...     

Tetrodotoxin--distribution and accumulation in aquatic organisms, and cases of human intoxication

Many pufferfish of the family Tetraodontidae possess a potent neurotoxin, tetrodotoxin (TTX). In marine pufferfish species, toxicity is generally high in the liver and ovary, whereas in brackish water and freshwater species, toxicity is higher in the skin. In 1964, the toxin of the California newt was identified as TTX as well, and since then TTX has been detected in a variety of other organisms. TTX is produced primarily by marine bacteria, and pufferfish accumulate TTX via the food chain that begins with these bacteria. Consequently, pufferfish become non-toxic when they are fed TTX-free diets in an environment in which the invasion of TTX-bearing organisms is completely shut off. Although some researchers claim that the TTX of amphibians is endogenous, we believe that it also has an exogenous origin, i.e., from organisms consumed as food. TTX-bearing animals are equipped with a high tolerance to TTX, and thus retain or accumulate TTX possibly as a biologic defense substance. There have been many cases of human intoxication due to the ingestion of TTX-bearing pufferfish, mainly in Japan, China, and Taiwan, and several victims have died. Several cases of TTX intoxication due to the ingestion of small gastropods, including some lethal cases, were recently reported in China and Taiwan, revealing a serious public health issue.     

The Tetrodotoxin Receptor of Voltage-Gated Sodium Channels—Perspectives from Interactions with μ-Conotoxins

Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide μ-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel’s voltage sensor. Most recently, the relatively small μ-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.