Cloning and sequence analysis of the phytoene synthase gene from a unicellular chlorophyte, Dunaliella salina

Phytoene synthase (Psy) catalyzing the dimerization of two molecules of geranylgeranyl pyrophosphate (GGPP) to phytoene may be rate limiting for the synthesis of beta-carotene in Dunaliella salina. To elucidate the carotenogenic pathway in D. salina, the complete Psy gene was first isolated by genome walking technology and suppression PCR. Subsequently, RT-PCR was performed to obtain the Psy cDNA of 1260 bp, which codes 420 amino acids. The Psy gene of total length of 2982 bp was found to consist of five exons and four introns when compared with the cDNA sequence. The D. salina Psy amino acid has a 78-89% similarity with many other higher plants, but a phylogenic analysis shows a closer relationship between the microalgal and cyanobacterial Psy.     

Carotenoid accumulation and characterization of cDNAs encoding phytoene synthase and phytoene desaturase in garlic (Allium sativum)

Phytoene synthase (PSY) and phytoene desaturase (PDS), which catalyze the first and second steps of the carotenoid biosynthetic pathway, respectively, are key enzymes for the accumulation of carotenoids in many plants. We isolated 2 partial cDNAs encoding PSY (AsPSY-1 and AsPSY-2) and a partial cDNA encoding PDS (AsPDS) from Allium sativum. They shared high sequence identity and conserved motifs with other orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of AsPSY1, AsPSY2, and AsPDS in the bulbils, scapes, leaves, stems, bulbs, and roots of garlic. High-performance liquid chromatography demonstrated that carotenoids were not biosynthesized in the underground organs (roots and bulbs), but were very abundant in the photosynthetic organs (leaves) of A. sativum. A significantly higher amount of β-carotene (73.44 μg·g(-1)) was detected in the leaves of A. sativum than in the other organs.     

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).     

加强Psy基因表达和通过RNAi抑制Lcy基因表达的载体构建

通过基因工程手段加强八氢番茄红素合成酶（Psy）基因的表达和通过RNAi抑制番茄红素β-环化酶（Lcy）基因（该基因是调控番茄红素转化为其他类胡萝卜素的主要的基因）的表达是培育高番茄红素品种的重要手段之一。根据GenBank中拟南芥的Psy基因序列（NM-121729），通过聚合酶链式反应（PCR）从拟南芥的cDNA中扩增出了长1296的Psy基因。根据GenBank中番茄的Lcy基因序列（X86452）和Pds启动子序列（U46919）分别设计特异引物从番茄中扩增出了Lcy基因的高度保守的长302bp的DNA片段和长1790的Pds启动子片段。根据RNAi的原理，将Lcy基因的长302bp的DNA片段以正反两个方向通过一段内含子序列连接在一起形成RNAi片段插入到抗Kan的pVCT2020的表达载体中，并通过具有果实特异性特点的启动子——八氢番茄红素去饱和酶基因（Pds）启动子控制该干扰片段的表达，同时将Psy基因在Pds启动子调控下插入到同一载体中，从而构建成了能同时加强Psy基因的表达和抑制Lcy基因表达的植物表达载体。     

Influence of daily collection and culture medium recycling on the growth and beta-carotene yield of Dunaliella salina

The halophilic green alga Dunaliella salina has the potential to be cultivated for beta-carotene-rich biomass, however, open-air systems need to be further improved in order to become more competitive and more economical, rather than leave the major beta-carotene consuming market derived from artificially synthesis. A set of daily collection ratios was designed and scaled up with the aim to harvest cell biomass and beta-carotene from D. salina at logarithmic phase; the yields were comparable to the normal culture without daily removal of culture. Daily collection of 1/7.5 volume of algal culture was found to be appropriate to keep the balance between the cell biomass and beta-carotene accumulation. Light intensity as one of the important factors would affect both cell growth and beta-carotene content synchronously. Further, the method of recycling 1/7.5 volume of culture after removal of algae cells was developed in order to decrease input cost for the effective production of beta-carotene, and both the resulting yields of the cell biomass and beta-carotene gained an advantage over those from the normal D. salina culture.     

Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.     

In vitro inhibition studies of phytoene desaturase by bleaching ketomorpholine derivatives

The herbicidal activities of homochiral steroisomeric 5-methy1-2-(3-trifluoromethybenzyl)-3-keto- morpholine derivatives were investigated in vitro as inhibitors of phytoene desaturase, a key enzyme in carotenoid biosynthesis. It was demonstrated that ketomorpholines are classical bleaching compounds which directly inhibit phytoene desaturase, accumulating phytoene at the expense of colored carotenoids. Ketomorpholines interact with phytoene desaturase in a noncompetitive manner with respect to phytoene. A structure-activity investigation for in vitro inhibition of phtoene desaturase activity revealed that the relative and absolute stereochemistry is important for optimum inhibition for the 5-methyl derivatives, and that the distance of the phenyl group from the ketomorpholine ring is critical for the inhibitory potential. The average herbicidal score on 7 weeds and the in vitro I(50) values related very well with the exception of two compounds. It was postulated that the discrepancies may possibly occur through modification in plants to compounds that are either more or less active herbicides.     

Spray-drying of the microalga Dunaliella salina: effects on beta-carotene content and isomer composition.

The effects of spray-drying of the unicellular microalga Dunaliella salina on its beta-carotene content and geometric isomer composition have been studied. The efficacy of a range of synthetic and natural antioxidants in preventing degradation of beta-carotene has been determined. Losses of beta-carotene and isomerization were minimal during processing for both the control (no exogenous antioxidants) and the samples containing butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ). However, the use of tocopherol-based antioxidants resulted in degradation of 52-72% of beta-carotene during the drying process. All dried powders of Dunaliella proved to be unstable during storage in the presence of light and air, with beta-carotene degraded according to a first-order kinetic model. Of the antioxidants studied, only TBHQ was successful in significantly minimizing degradation (degradation constants of 0.03 and 0.04 days(-)(1), compared to 0.53 days(-)(1) for the respective control). For control powders and those with BHT added to the feed, the degradation constants were reduced to values between 0.27 and 0.37 days(-)(1) by restricting light and flushing with nitrogen; however, storage in the dark alone had no effect. For more slowly degrading powders having TBHQ added to the feed, it was clear that degradation of beta-carotene was influenced by both light and oxygen. During storage the 9-cis isomer of beta-carotene was significantly more unstable than the all-trans form. TBHQ was, however, successful in reducing relative losses of this isomer for samples stored in the dark. The results suggest a dominant photodegradative mechanism for the loss of the 9-cis isomer of beta-carotene.     

Beta-carotene isomer composition of sub- and supercritical carbon dioxide extracts. Antioxidant activity measurement

In the present work sub- and supercritical extraction conditions using carbon dioxide were studied in order to obtain extracts with different compositions from the green microalgae Dunaliella salina. Different compositions of beta-carotene isomers were identified in the extracts by using HPLC-DAD. Also, antioxidant activity of the extracts was measured using a TEAC assay. An experimental design was applied considering two factors, extraction pressure and temperature, in a wide range of values, trying to maximize the extraction yield. Higher yields were obtained at high pressures and low temperatures, that is, at higher CO2 densities. Attempts were made to correlate the antioxidant activity of the extracts with their chemical composition by means of principal component analysis. A certain relationship was found between their antioxidant activity and the isomeric composition of beta-carotenes. As a result, an original equation is proposed to predict the antioxidant activity of extracts from D. salina in terms of the ratio 9-cis-beta-carotene/all-trans-beta-carotene, the concentration of alpha-carotene, and, especially, the concentration of 9-cis-beta-carotene.     

Carotenoid biosynthetic pathway in the citrus genus: number of copies and phylogenetic diversity of seven genes

The first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecific diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, and Zep genes labeled with [alpha-(32)P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecular markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the results of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumulation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci.     

Relationship of carotenoids and tocopherols in a sample of carrot root-color accessions and carrot germplasm carrying Rp and rp alleles

Carotenoids and tocopherols are powerful antioxidants synthesized in plants from a common precursor. They may offer significant health benefits to humans. Seed oils have been shown to possess high levels of tocopherols, but little is known about their levels in the edible portions of most vegetable crops. A two-year field experiment was conducted at two locations to assess levels of major carotenoids and tocopherols in carrot (Daucus carota) root and leaf tissue. Levels of compounds in root tissue reported on a dry weight basis were as follows: alpha-tocopherol, 0.04-0.18 ppm; lycopene, 0.00-52.94 ppm; alpha-carotene, 10.63-1504.76 ppm; and beta-carotene, 26.69-1673.76 ppm. Higher levels of all carotenoids were measured in phloem tissue than in xylem. Leaf tissue levels of tocopherols measured on a dry weight basis ranged from 0.02 to 0.85 ppm, whereas levels of carotenoids ranged from 12.81 to 411.66 ppm. In xylem tissue, alpha-tocopherol was significantly (P < or =0.001) positively correlated with alpha-carotene (r = 0.65) and with beta-carotene (r = 0.52). This positive correlation indicates it may be possible to select for both increased alpha-tocopherol and carotenoids in carrot. The reduced pigment (rp) mutation of carrot exhibited a 96% reduction in levels of alpha- and beta-carotene and a 25-43% reduction in alpha-tocopherol when compared to a near-isogenic line. In plants homozygous for rp, a substantial increase was observed in phytoene, a precursor to carotenoids, suggesting the location of the rp lesion in the carotenoid synthesis pathway.     

巴西橡胶树HbMCS1基因的克隆及表达分析

2C-甲基-D-赤藓糖醇-2,4-环化磷酸合成酶（2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,MCS）是异戊烯基焦磷酸合成途径之一——甲基赤藓糖磷酸（methylerythritol phosphate,MEP）途径中的第五个酶,催化2-磷酸-4- (胞苷-5’-二磷酸)- 2-C-甲基-D-赤藓糖醇生成2-C-甲基-D-赤藓糖醇-2-4-环化磷酸。根据植物MCS的同源序列设计引物,通过RT-PCR结合RACE的方法在橡胶树中获得了与其相应的MCS基因,命名为HbMCS1。序列分析表明HbMCS1长965bp,编码241个氨基酸,该氨基酸序列与长春花、拟南芥、水稻、银杏和三尖杉的MCS同源性分别达到70.8%、69.4%、64.9%和63.3%和62.2%。半定量RT-PCR结果显示,伤害诱导胶乳HbMCS1的表达,乙烯对HbMCS1的表达几乎没有影响;HbMCS1的表达具有组织差异性,在愈伤组织中大量表达,在叶片和胶乳中微量表达。HbMCS1的克隆和表达分析为了解MEP途径在橡胶树胶乳中的作用和对天然橡胶生物合成的调控作用打下了基础。     

编码日本梨树(Pyrus pyrifolia )花粉类β-1,3-葡聚糖酶蛋白的cDNA克隆与核苷酸序列

构建了日本梨树(Pyrus pyrifolia)花粉cDNA文库,发现了一个编码花粉管分泌的类β-1,3-葡聚糖酶蛋白(BGN-1)的cDNA(bgn-1)并测定了其序列.bgn-1由1408 bp组成,包括47 bp的5'-非翻译区,由1194bp组成并编码了397个氨基酸的ORF和167 bp的3'-非翻译区.3'-非翻译区含有1个NUE(AATTAA)和3个似FUE的基序,分别位于1369～1374(AATTAA)、1320～1325、1327～1332(TTTTTA)和1363～1368(TTTGGA).bgn-1编码的蛋白(BGN-1)与DDBJ中的植物β-1,3-葡聚糖酶的序列相似性范围为37%～59%,与3D结构已知的大麦β-1,3-葡聚糖酶(GⅡ)的序列相似性为39.7%.对GⅡ与BGN-1进行的疏水簇分析(HCA同源分为87.1%,＞75%的一般标准)和使用3D-PSSM软件进行的分析暗示,BGN-1的3D结构与大麦GⅡ同源性最大(≥95%的肯定度).BGN-1的信号肽长度为22 aa,成熟的BGN-1(375 aa)的理论分子量为4 0723 D,理论pI值为9.59,诊断氨基酸残基模式(D,L,S,L)与禾谷类的与生长相关的亚家族D的诊断氨基酸残基模式(D,L,S,Q)极相似.在BGN-1的C-端延伸区的位点352～367之间发现了1个功能不清,重复出现的ProXXPro结构.     

Carbon and hydrogen stable isotope ratios of carotenoids and beta-carotene-based dietary supplements

Considering the increasing nutritional and commercial importance of carotenoids, there is an interest in developing a reliable method for authenticity assessment of these compounds. Applying isotope ratio mass spectrometry using elemental analysis in the "combustion" (C) and "pyrolysis" (P) modes (EA-C/P-IRMS), the delta (13)C V-PDB and delta (2)H V-SMOW values of selected carotenoids and alpha/beta-carotene-based commercial dietary supplements were determined in comparison to those of synthetic and "natural" references. The delta (13)C V-PDB and delta (2)H V-SMOW values of synthetic beta-carotene samples ( n = 4), ranging from -25.3 per thousand to -26.4 per thousand and from -144 per thousand to -155 per thousand, respectively, differed clearly from the data determined for carotenoids from various natural sources, including C 3 plant material ( n = 9; delta (13)C V-PDB ranging from -28.5 per thousand to -32.8 per thousand and delta (2)H V-SMOW from -180 per thousand to -275 per thousand) and microalgae Dunaliella salina ( n = 1; delta (13)C V-PDB value of -15.6 per thousand and delta (2)H V-SMOW value of -191 per thousand). From five commercial dietary supplements under study, two revealed delta (13)C V-PDB and delta (2)H V-SMOW values in areas as found for synthetic references, and the other three had values near those of biotechnological beta-carotene produced by D. salina. The delta (13)C V-PDB and delta (2)H V-SMOW values recorded for natural lycopene ( n = 4) and lutein ( n = 5) ranged from -31.1 per thousand to -31.8 per thousand and from -180 to -201 per thousand, as well as from -28.8 per thousand to -32.2 per thousand and from -186 per thousand to -245 per thousand, respectively. Synthetic canthaxanthin ( n = 3) exhibited delta (13)C V-PDB and delta (2)H V-SMOW values ranging from -25.0 per thousand to -28.6 per thousand and from -133 per thousand to -153 per thousand, respectively. The EA-C/P-IRMS application of this study showed that the natural stable isotopic composition of carotenoids is a powerful tool for determining their origin.     

DIG和碱性磷酸酶标记cDNA探针检测番木瓜PRSV的比较

利用地高辛(DIG)标记和碱性磷酸酶直接标记的cDNA探针核酸分子杂交及RT－PCR方法对番木瓜环斑病毒(PRSV)进行了检测。根据Genebank发表的PRSV中国Sm株系的外壳蛋白基因序列，设计特异引物，以美中红（Carica papaya L. cv. Meizhonghong ）带病样品的RNA为模板进行RT-PCR扩增反应,将其目的cDNA片段克隆到pGEM-T easy 质粒载体上，并测序。以重组质粒作模板，用PCR－DIG标记方法制备cDNA探针，另一种非放射性探针是经凝胶电泳分离、纯化cDNA片段，用碱性磷酸酶直接进行标记。结果表明，(1)美中红No.2序列与中国优势株系Sm的同源性为94.7%；(2) DIG标记的三种cDNA探针（861bp，455bp和215bp）对样品的总RNA检测结果是一致的, 且861bp的探针杂交斑点最为清晰，上述核酸杂交结果与RT－PCR检测结果相符, 核酸分子杂交检测的灵敏度和特异性能满足常规检测的需要；(3)碱性磷酸酶直接标记861bp的cDNA探针可进行PRSV的斑点杂交检测，而455bp的cDNA片段用碱性磷酸酶直接标记时，不能获得有效的杂交结果；(4)本试验还用DIG标记探针对叶脉进行了印迹杂交检测,结果与RT－PCR检测结果相符。     

番茄ACO基因的克隆及其RNAi对乙烯产量的抑制

筛选番茄幼苗cDNA文库获得与番茄抗性相关的克隆E11，设计引物，利用RT-PCR方法从受到病原浸染的番茄叶片中获得长1018 bp的候选片段，同源性分析发现该片段与其他作物上已发表的ACO基因序列高度同源：同源率从83%~99%，推断该基因为番茄ACO基因家族的新成员。在此基础上，用BP克隆的方法构建该基因的RNA干涉（RNAi）载体pD311，对番茄进行遗传转化，获得卡那霉素抗性植株27棵，分子检测证实外源片段成功导入其中番茄基因组中。对获得的转基因植株的乙烯生成量测定结果表明，RNAi结构的导入大大抑制了内源ACO基因的表达，从而导致乙烯的生成大大降低。     

Digestive Stability, micellarization, and uptake of beta-carotene isomers by Caco-2 human intestinal cells

While isomeric profiles of carotenoids found in food often differ from those in body fluids and tissues, insights about the basis for these differences remain limited. We investigated the digestive stability, relative efficiency of micellarization, and cellular accumulation of trans and cis isomers of beta-carotene (BC) using an in vitro digestion procedure coupled with human intestinal (Caco-2) cells. A meal containing applesauce, corn oil, and either water-soluble beadlets (WSB) or Dunaliella salina (DS) as a BC source was subjected to simulated gastric and small intestinal digestion. BC isomers were stable during digestion, and the efficiency of micellarization of cis-BC isomers exceeded that of all-trans-BC isomers. The cellular profile of carotenoids generally reflected that in micelles generated during digestion, and intracellular isomerization was minimal. These data suggest that cis isomers of BC are preferentially micellarized during digestion and transferred across the brush-border surface of the enterocyte from mixed micelles with similar efficiency as all-trans-BC at the concentrations of the carotenoids utilized in this study.     

番茄ACO基因的克隆及其RNAi对乙烯释放的抑制

筛选番茄(Lycopersicon esculentum)幼苗cDNA文库获得与番茄抗性相关的克隆E11,设计引物,利用RT-PCR方法从受到病原侵染的番茄叶片中获得长1018bp的候选片段,同源性分析发现该片段与其它作物上发表的ACO基因序列高度同源,同源率83%～99%,推断该基因为番茄ACO基因家族的新成员。在此基础上,用BP克隆的方法构建该基因的RNA干涉(RNAi)载体pD311,对番茄进行遗传转化,获得卡那霉素抗性植株27棵,分子检测证实外源片段成功导入番茄基因组中。对获得的转基因植株的乙烯生成量测定结果表明,RNAi结构的导入大大抑制了内源ACO基因的表达,从而导致乙烯的生成大大降低。