Comparison of selenium treatments of crops in the field

Field experiments with spring and winter barley and ryegrass were carried out to compare the effect of fertilizers enriched with selenate or selenite with foliar application on the selenium (Se) concentrations in the crops. Application of about 20 g Se/ha given as selenate or about 100 g as selenite in a PK fertilizer and about 5 g Se/ha sprayed on the plants increased the Se-concentrations from insufficient levels to levels that met the animal nutritional requirement. Ryegrass obtained sufficient Se concentrations at lower levels of added Se than did the barley. Toxic concentrations did not occur. The choice of method thus depends on the farming practice in the individual cases.     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

A perennial ryegrass CBF gene cluster is located in a region predicted by conserved synteny between Poaceae species

CBF/DREB1 proteins are the most important regulators of the cold temperature signaling pathway in many plants. CBF genes are candidates for low-temperature tolerance QTL in wheat and barley. Ten novel putative CBF cDNAs of perennial ryegrass (Lolium perenne L.) have been isolated from cold-treated leaf tissue. Their primary structures contain some conserved motifs, characteristic of the gene class. Phylogenetic analysis revealed that LpCBF genes were attributable to the HvCBF3-, and HvCBF4-subgroups following the previously proposed classification of barley CBF genes. RT-PCR analysis revealed that the expression of LpCBF genes was rapidly induced in response to low temperature and that the expression pattern under the low-temperature conditions for a long period was different between the various LpCBF genes. Five of the ten LpCBF genes were assigned to the genetic linkage map using the p150/112 reference mapping population. LpCBFIb, LpCBFII, LpCBFIIIb and LpCBFIIIc were mapped on LG5 forming a cluster within 2.2 cM, while LpCBFVb was located on LG1. Based on comparative genetic studies, conserved synteny for CBF gene family was observed between the Triticeae cereals and perennial ryegrass. Information on the perennial ryegrass CBF genes at both the molecular and genetic level obtained in this study would be useful for the further study on the role of CBF genes and low-temperature tolerance in grasses.     

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne)

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.     

饲料硒含量对黄颡鱼肠系膜脂肪组织中脂类代谢和miRNAs表达水平的影响

使用3种不同硒含量的饲料饲养黄颡鱼12周, 研究饲料硒含量对黄颡鱼(Pelteobagrus fulvidraco)肠系膜脂肪组织脂类代谢和miRNAs表达水平的影响, 饲料硒含量分别为0.03(低硒组)、0.25(适宜硒组)和6.39 mg Se/kg饲料(高硒组)。结果表明, 相比较于适宜硒组, 高硒和低硒组中甘油三酯(TG)含量显著升高(P<0.05), 葡萄糖-6-磷酸脱氢酶(G6PD)和6-磷酸葡萄糖脱氢酶(6PGD)的酶活性在低硒组中活性显著下降(P<0.05), 在高硒组中活性显著上升(P<0.05); 异柠檬酸脱氢酶(ICDH)的酶活性在低硒组中没有显著性变化(P>0.05), 在高硒组中显著下降(P<0.05); 苹果酸酶(ME)和脂肪酸合成酶(FAS)的酶活性在低硒和高硒组中均显著上升(P<0.05)。相比于适宜硒组, 在高硒组miR-26a、miR-183、miR-135、let-7b、let-7c和let-7g的表达量显著上升(P<0.05), 而在低硒组中没有显著性差异(P>0.05); miR-181a-5p和let-7e在低硒组中显著上升(P<0.05), 在高硒组中没有显著性差异(P>0.05); miR-130和miR-203a在低硒和高硒组中表达量均显著上升(P<0.05); miR-200a、miR-143、let-7d和let-7f在低硒组中表达量显著下降同时在高硒组中表达量上升(P<0.05)。miR-143、miR-203a和miR-130在脂肪组织中高表达, 且对硒产生强响应, 通过TargetScanFish6.2和miRwalk3.0共预测3个miRNA的靶基因, 并对靶基因进行KEGG富集分析, 结果显示, miR-143的靶基因在矿物盐吸收、不饱和脂肪酸生物合成、胰岛素通路、自噬、脂肪酸代谢、鞘脂类代谢、调节脂肪细胞脂肪分解和甘油磷脂代谢等途径中显著富集(P<0.05, FDR≤0.05); miR-203a的靶基因在胰岛素抵抗、胰岛素通路、调节脂肪细胞脂肪分解和自噬等代谢途径、信号通路中显著富集(P<0.05, FDR≤0.05); miR-130的靶基因在胰岛素通路、胰岛素抵抗、自噬、鞘脂类代谢、调节脂肪细胞脂肪分解和mTOR通路中显著富集(P<0.05, FDR≤0.05)。综合分析发现, miR-143、miR-203a和miR-130的靶基因共同富集在脂肪酸合成、调节脂肪细胞脂肪分解、胰岛素抵抗等脂代谢相关的通路中, 选取共富集通路中的靶基因进行qPCR检测, 结果显示fas、碳水化合物反应元件结合蛋白α(chrebpα)、肉碱棕榈酰转移酶1α(cpt1α)、激素敏感酯酶(hsl)、固醇调节元件结合蛋白(srebp1)、过氧化物酶体增殖剂激活受体α(pparα)和脂肪组织甘油三酯酶(atgl)的表达趋势与miR-143、miR-203a和miR-130的表达趋势相符合, 推测饲料硒缺乏和过量诱导黄颡鱼肠系膜脂肪组织脂质沉积是通过诱导miR-143、miR-203a和miR-130的表达量上调, 进而负调控靶基因chrebpa、cpt1a、hsl、srebp1、ppara和atgl的表达实现。     

Expressed sequence tags and chromosomal localization of cDNA clones from a subtracted retinal pigment epithelium library.

Expressed sequence tags (ESTs) provide useful molecular landmarks for physical mapping and identify the position of an expressed region in the genome. The use of subtracted cDNA libraries enriched for tissue-specific genes as a source of ESTs should reduce the repetitive isolation of constitutively expressed sequences. We report here the sequence tags from the 3'-end region of 58 new directionally cloned cDNAs from a subtracted human retinal pigment epithelium (RPE) cell line library. Eight of the cDNAs have been assigned to human chromosomes using PCR-based EST assays. Chromosomal mapping of subtracted RPE cDNA clones may also help in identifying candidate genes for inherited eye diseases.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

A plant plasma membrane ATP binding cassette-type transporter is involved in antifungal terpenoid secretion

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense.     

混播草地不同种群再生性的研究

在不同刈割频率和时间尺度下 ,对混播草地多年生黑麦草 (Lolium perenne)分蘖数和叶片生长、白三叶 (Trifoliumrepens)分枝数和匍匐茎生长及不同种群年产量和组分进行了连续 3年的监测研究 .结果表明 ,刈割能刺激黑麦草叶片、白三叶匍匐茎生长和分枝数发生 ,保持混播草地黑麦草和白三叶的适宜比和稳定共存 ,提高草地年生产力 ,但不同刈割频率和刈割时间对其影响差异不显著 (P >0 .0 5 ) .黑麦草叶片生长对 6月刈割效果比 8月明显 ,而白三叶匍匐茎生长则与之相反 ,黑麦草产量主要取决于叶片生长 ,白三叶产量主要取决于匍匐茎分枝数 .刈割的黑麦草、白三叶产量组分比分别为 5 0 %、15 % ,比试验前约低 10 %、5 % ,而CK为 39%、6 % .     

Selenium improves the antioxidant ability against aluminium-induced oxidative stress in ryegrass roots

We investigated the role of selenium (Se) against aluminium (Al) stress in ryegrass by evaluating the growth responses and the antioxidant properties of plants cultured hydroponically with Al (0 or 0.2 mM) and selenite (0–10 µM Se). Al addition significantly reduced the yield and length of shoots and roots, and most Al was accumulated in the roots. Al also enhanced lipid peroxidation and activated the peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD) enzymes in the roots. Se application up to 2 µM improved root growth and steadily decreased thiobarbituric acid reactive substances (TBARS) accumulation in plants treated with 0 and 0.2 mM Al. However, above 2 µM, Se induced stress in plants grown with or without Al. Significant changes in antioxidant enzymes activities were also found as a result of the added Se. At low Se addition levels POD was activated, whereas APX activity decreased irrespective of added Al. Furthermore, Se supplied up to 2 µM greatly decreased root SOD activity in Al-stressed plants. Our study provides evidence that Se alleviated the Al-induced oxidative stress in ryegrass roots through the enhancement of the spontaneous dismutation of superoxide radicals and the subsequent activation of POD enzyme.     

Variation in selenium tolerance and accumulation among 19 Arabidopsis thaliana accessions

Selenium (Se) is an essential element for many organisms but also toxic at higher levels. The objective of this study was to identify accessions from the model species Arabidopsis thaliana that differ in Se tolerance and accumulation. Nineteen Arabidopsis accessions were grown from seed on agar medium with or without selenate (50 microM) or selenite (20 microM), followed by analysis of Se tolerance and accumulation. Tissue sulfur levels were also compared. The Se Tolerance Index (root length+Se/root length control) varied among the accessions from 0.11 to 0.44 for selenite and from 0.05 to 0.24 for selenate. When treated with selenite, the accessions differed by two-fold in shoot Se concentration (up to 250 mgkg(-1)) and three-fold in root Se concentration (up to 1000 mgkg(-1)). Selenium accumulation from selenate varied 1.7-fold in shoot (up to 1000 mgkg(-1)) and two-fold in root (up to 650 mgkg(-1)). Across all accessions, a strong correlation was observed between Se and S concentration in both shoot and root under selenate treatment, and in roots of selenite-treated plants. Shoot Se accumulation from selenate and selenite were also correlated. There was no correlation between Se tolerance and accumulation, either for selenate or selenite. The F(1) offspring from a cross between the extreme selenate-sensitive Dijon G and the extreme selenate-tolerant Estland accessions showed intermediate selenate tolerance. In contrast, the F(1) offspring from a cross between selenite-sensitive and -tolerant accessions (Dijon GxCol-PRL) were selenite tolerant. The results from this study give new insight into the mechanisms of plant selenium (Se) tolerance and accumulation, which may help develop better plants for selenium phytoremediation or as fortified foods.     

Expression of a mouse selenocysteine lyase in Brassica juncea chloroplasts affects selenium tolerance and accumulation

Selenium is an essential nutrient for many organisms, as part of certain selenoproteins. However, selenium is toxic at high levels, which is thought to be due to non-specific replacement of cysteine by selenocysteine leading to disruption of protein function. In an attempt to prevent non-specific incorporation of selenocysteine into proteins and to possibly enhance plant selenium tolerance and accumulation, a mouse selenocysteine lyase was expressed in Brassica juncea (Indian mustard) chloroplasts, the site of selenocysteine synthesis. This selenocysteine lyase specifically breaks down selenocysteine into elemental selenium and alanine. The transgenic cpSL plants showed normal growth under standard conditions. Selenocysteine lyase activity in the cpSL transgenics was up to 6-fold higher than in wild-type plants. The cpSL transgenics contained up to 40% less selenium in protein compared to wild-type plants, indicating that Se flow in the plant was successfully redirected. Surprisingly, the selenium tolerance of the transgenic cpSL plants was reduced, perhaps due to interference of produced elemental selenium with chloroplastic sulphur metabolism. Shoot selenium levels were enhanced up to 50% in the cpSL transgenics, but only during the seedling stage.