RS基因转化胡萝卜及高白藜芦醇转基因胡萝卜株系筛选

摘要：白藜芦醇是具有抗病及多种保健功能的芪类次生代谢物,胡萝卜则是富含β胡萝卜素等多种营养成分的重要蔬菜。本研究着眼于创建具有白藜芦醇保健功能的胡萝卜新种质资源，在对葡萄白藜芦醇合成酶（Resveratrol synthase 简称RS）基因针对在胡萝卜中表达的密码子优化基础上构建无潮霉素抗性基因的转化载体pCB－RS－hyg-，应用农杆菌介导遗传转化方法将pCB和pCB－RS－hyg-共转化到胡萝卜并获得再生植株。经过T0、T1和T2代的逐代筛选，获得2个白藜芦醇含量较高的转基因胡萝卜株系，为进一步开发利用以及相关代谢途径的分子生物学研究奠定了重要基础。     

植酸酶根特异表达载体的构建及大豆转化

应用PCR方法分别从胡萝卜基因组中扩增出96bp的外展蛋白信号肽编码序列片段,从拟南芥基因组中扩增出1454bp的pyk10启动子片段,用RT-PCR方法从无花果曲霉（Aspergillus ficuum3.4322）中扩增出phyA基因,长1347bp。然后,分别克隆到pMD18-T载体。应用已设计的限制酶切位点,通...     

STAT3基因转染牛乳腺上皮细胞及生物学特性观察

应用PCR技术从含有人STAT3基因cDNA的质粒pOTB7中扩增STAT3基因片段。将其克隆入真核表达载体pEGFP-C1中,构建重组表达质粒。利用脂质体介导法将重组表达质粒转染原代牛乳腺上皮细胞(Bovine Mammary Epithelial cells)。G418筛选阳性克隆,RT-PCR检测STAT3基因在牛乳腺上皮细胞中的表达情况,并通过流式细胞术(Flow cytometry)检测转染细胞的增殖能力、周期以及DNA含量。结果表明,转染24h后大约16%的细胞被导入含STAT3基因的重组表达质粒。在mRNA水平证实细胞内有STAT3基因的高表达。导入STAT3基因后,细胞增殖能力增强,染色体倍数发生变化,细胞的增殖寿命较对照细胞延长了13代,表明导入STAT3基因后,能延长体外培养的牛乳腺上皮细胞的寿命。     

Untersuchungen über den Einfluß des Kinetins und des Eisens auf den Nukleinsäure- und Proteinstoffwechsel von Karottengewebekulturen

The Influence of Kinetin and Iron on Nucleic Acid and Protein Metabolism of Carrot Tissue Cultures Carrot-root tissues cultured in a nutrient solution containing Kinetin had a high rate of cell division, that led to an undifferentiated callus, and a relatively low DNA content per cell. The same tissues growing without Kinetin had a smaller rate of cell division and a higher DNA content, and showed the ability to form roots. By addition of ³²P to the nutrient solution, the cultures growing with Kinetin showed a higher specific activity of DNA than those growing without Kinetin. From previous experiments and results of other workers the possibility of the occurence of a metabolic labile DNA fraction in the cultures which grew without Kinetin is discussed. A comparison between the influence of iron and Kinetin on cell division activity, root formation, DNA, RNA and amino acid content of the tissue cultures was made on a cell basis. Iron deficiency or absence of Kinetin in the nutrient solution reduced the cell division activity of carrot tissue cultures and increased the content of DNA, RNA and soluble amino acids. The protein content was reduced by iron deficiency but was increased by the absence of Kinetin. Cultures, growing without Kinetin were able to form roots, while those, growing in an iron deficient medium were unable to show the latter phenomenon.     

Effect of intrinsic and extrinsic factors on the interaction of plant pectin methylesterase and its proteinaceous inhibitor from kiwi fruit

A proteinaceous pectin methylesterase inhibitor (PMEI) was isolated from kiwi fruit (Actinidia chinensiscv. Hayward) and purified by affinity chromatography on a cyanogen bromide (CNBr) Sepharose 4B-orange PME column. The optimal pH of banana PME activity was 7.0, whereas that for carrot and strawberry PME activity was 9.0. The optimal pH for the binding between kiwi fruit PMEI and these PMEs was 7.0. The kiwi fruit PMEI has a different affinity for PME depending on the plant source. The inhibition kinetics of kiwi fruit PMEI to banana and strawberry PME followed a noncompetitive type, whereas that to carrot PME followed a competitive type. The kiwi fruit PMEI was mixed with banana, carrot, and strawberry PME to obtain PMEI-PME complexes, which were then subjected to thermal (40-80 degrees C, atmospheric pressure) or high-pressure (10 degrees C, 100-600 MPa) treatment. Experimental data showed that the PMEI-PME complexes were easily dissociated by both thermal and high-pressure treatments.     

穿刺协同乙醇预处理对胡萝卜片热风干燥特性影响

干燥效率低导致的能耗高、品质劣变是果蔬热风干燥加工面临的严峻挑战。为改善胡萝卜热风干燥特性,该研究以横切和纵切方式获得的胡萝卜片为研究材料,对其进行穿刺（perforation pretreatment,PT）、乙醇浸渍（alcohol pretreatment,AT）和穿刺协同乙醇浸渍预处理（perforation synergistic alcohol pretreatment,PAT）。研究发现：同未预处理的胡萝卜片相比,PT、AT和PAT预处理技术的应用均显著提高了胡萝卜有效水分扩散系数及热风干燥速率,并将干燥时间缩短10.61%～50.00%,其中PAT样品干燥时间最短。相同预处理方式下,纵切的胡萝卜片相比于横切表现出较高的有效水分扩散系数、较快的干燥速率及较短的干燥时间。进一步,我们提出了PAT预处理具有较高干燥效率的“三重奏机制”：预脱水降低干燥负荷、毛细管流促进水分传质、细胞通透性的增强降低水分传质阻力。在品质方面,PAT预处理显著改善了纵切胡萝卜片的体积收缩、复水性、色泽、总类胡萝卜素和抗坏血酸含量、抗氧化活性及风味等品质特性。因此,为高效获得品质相对较优的脱水胡萝卜,可在其纵向切片后进行PAT预处理。该研究为改善胡萝卜热风干燥提供了一种简单有效的预处理技术,同时也为其他果蔬提质、增效、节能干燥提供参考。     

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.     

一个新的马铃薯patatin classⅠ基因的cDNA在烟草中的表达及其酶活性

摘要：将马铃薯(Solanum tuberosum )块茎特异蛋白patatin classⅠ基因cDNA与CaMV 35S启动子融合，构建了植物表达载体pBSSP。用电激法将表达载体导入根癌农杆菌（Agrobacterium tumefaciens）LBA4404，然后转化烟草(Nicotiana tabacum )叶片获得再生植株。经PCR、PCR-Southern、Northern杂交和蛋白质检测，证明patatin classⅠ基因cDNA已整合到烟草基因组中并在转基因植株中转录和表达。酯酰水解酶（lipid acylhydrolase, LAH）活性分析显示，转基因烟草植株的叶片中LAH活性明显提高。     

胡萝卜悬浮细胞原生质体的培养研究

继代培养 4～ 5d后的胡萝卜悬浮细胞 ,经酶解获得大量原生质体 ,并在不同的培养基上培养 ,探讨了影响原生质体培养的多种因素。实验表明 :①原生质体的培养密度在 5× 1 0 4 mL～ 5× 1 0 5 mL范围内均可 ,以 2 5× 1 0 5 mL为佳 ;②对于细胞的分裂、细胞团的生长 ,以改良的B5培养最好 ,N6培养其次 ,MS较差 ;③适当降低原生质体培养基中的甘露醇浓度 ,有利于细胞的分裂和生长 ;④在本实验所涉及的培养方法之中 ,以液体浅层培养为优。     

Sucrose transporter NtSUT1 confers aluminum tolerance on cultured cells of tobacco (Nicotiana tabacum L.)

The role of plasma membrane-localized sucrose transporter (NtSUT1) was investigated using cultured tobacco cell (Nicotiana tabacum L.) line BY-2. The wild type (WT) cells were first transformed with the NtSUT1 gene or its fragments cloned from tobacco cell line SL to form the over-expression (OX) and suppression (RNAi) cell lines, respectively. Using OX and RNAi transgenics, the role of NtSUT1 in growth capacity of actively growing cells and in aluminum (Al)-treated cells was examined. During the logarithmic phase of growth in nutrient medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), both the rate of sucrose uptake measured with radio-tracer and the content of soluble sugars were higher in OX and lower in RNAi cell lines compared to WT. Overall, the content of soluble sugars negatively correlated with the time necessary for doubling mass (fresh weight). When cells were treated without (control) or with Al in a simple medium containing calcium, sucrose and 2-(N-morpholino)ethanesulfonic acid (MES; pH 5.0) for up to 18 h, the expression of NtSUT1 under its native promoter, or under the control of strong constitutive cauliflower mosaic virus (CaMV) 35S promoter, was strongly dependent on the presence of 2,4-D. Thereafter, the cells were preferentially treated in the presence of 2,4-D. During 6 h after a start of the control treatment, sucrose uptake rates were, compared to WT, slightly higher and lower in OX and RNAi lines respectively. The addition of Al reduced the sucrose uptake rates of OX and WT to the level of RNAi line, indicating that Al inhibits sucrose uptake via NtSUT1. During the post-Al culture of control and Al-treated cells in a nutrient medium, sucrose uptake rates were much higher in OX compared to WT and RNAi lines, which closely and positively correlated with the growth capacity of the cells. Judging from the growth capacity of Al-treated cells relative to that of control cells, OX cells were more tolerant to Al than WT and RNAi. In summary, we conclude that over-expression of NtSUT1 confers higher growth capacity in actively growing cells as well as in Al-treated cells.     

Einfluß des Belastungsgrades unterschiedlicher Böden an ausgewählten Pestizid wirkenden Chlorkohlenwasserstoffen auf deren Aufnahme durch Möhren im Gefäßversuch

Influence of contamination of selected, pesticid effective hydrocarbons on their uptake by carrots in a pot experiment Exemplary soils from low up to higher grades of contamination with selected pesticid effective hydrocarbons (CHC) were used in a pot experiment on the uptake of these xenobiotica by carrot plants. Carrots are able to take up pesticid effective chlorinated hydrocarbons which was expected because of their oil cells and the lipophilic character of the examined substances. There is a correspondingly higher plant uptake from soils with a higher load of pollutants as from soils with a lower grade of contamination. Despite the high concentrations of beta- and alpha-HCH in the plants from the site Muldenaue (mg/kg dm] the total uptake per pot was relatively small (below 2 mg/pot). The transferfactors soil/plant show in all plant parts no enrichment of organochlorpesticides (foliage 0.021 < carrot body 0.156 < fine roots 0.975).     

不同水分条件下磷肥运筹对小麦旗叶和穗部叶绿素及核酸含量的影响

[目的]在干旱和半干旱地区,缺磷常导致作物产量下降.研究不同水分状况下,磷素施用次数对花后小麦旗叶和穗部维持光合效能及胚乳细胞分裂能力的影响,为科学施用磷肥提供理论依据.[方法]以冬小麦品种'新冬23号'和'新冬20号'为试验材料开展裂区田间试验.设干旱胁迫(DT,灌水量为5625 m3/hm2)和适水灌溉(WT,灌水...     

过量表达盐芥TsIPK2基因增强转基因水稻耐盐性

【目的】本试验以野生型(WT)和转盐芥TsIPK2基因的水稻为材料,研究NaCl胁迫条件下过量表达TsIPK2基因对水稻植株抗盐胁迫能力的影响。【方法】取水稻材料种子和其3叶龄幼苗,分别在不同NaCl浓度(0、50、100、150、200 mmol/L)下进行处理。检测WT与过量表达TsIPK2基因水稻种子的发芽率、主根长和芽长、幼苗的丙二醛(MDA)和脯氨酸的含量,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,以及与胁迫相关的5个基因的表达。【结果】在盐胁迫下,与野生型相比,转基因水稻具有更好的发芽率、主根长和芽长。野生型和转基因水稻两者的脯氨酸含量增加,转基因水稻的积累量显著高于野生型,但是转基因水稻MDA含量增加幅度小于野生型。野生型和转基因水稻幼苗SOD酶活性均增加,但转基因植株的酶活性显著高于野生型;二者POD酶活性呈现先升高后下降的趋势,但是二者活性没有显著的差别;转基因水稻的CAT活性也呈现先升高后下降的趋势,然而野生型水稻的CAT活性在盐胁迫下没有显著的变化。高盐处理后,野生型和转基因水稻的5个与胁迫相关的基因表达倍数都增加,与野生型相比,转基因水稻的OsP5CS1、OsSOD、OsCATB和OsLEA3的表达量显著升高,而OsPOX1基因的表达量变化不显著。【结论】过量表达TsIPK2基因能够通过增强水稻的渗透调节能力、抗氧化胁迫能力并调节胁迫相关基因的表达,以提高水稻的耐盐性。     

鸡γ-干扰素基因在昆虫细胞/杆状病毒载体系统中的表达

摘要：将鸡（Gallus gallus）的γ-干扰素（interferon-γ, IFN-γ）基因亚克隆到杆状病毒转移载体pMelBacB的BamHⅠ位点上，构建真核转移载体pMelBacB-ChIFN-γ，经限制性内切酶消化、ChIFN-γ特异引物PCR鉴定和序列测定，证明目的基因正确克隆到载体的预期位点。将纯化的pMelBacB-ChIFN-γ质粒与杆状病毒DNA（Bac-N-BlueTM DNA）共转染sf9昆虫细胞，经3轮蓝斑筛选纯化，获得了重组杆状病毒rBaculovirus-ChIFN-γ。提取病毒DNA经ChIFN-γ特异引物和重组杆状病毒特异引物PCR鉴定，证明获得了纯化的重组杆状病毒，命名为rBaculovirus-ChIFN-γ。用该重组病毒接种sf9昆虫细胞，收获接种后不同时间的细胞进行SDS-PAGE,结果表明ChIFN-γ基因在昆虫细胞中获得了表达，表达的重组蛋白分子量约为19 kD。应用在大肠杆菌(Escherichia coli )原核表达系统中表达的重组蛋白制备的兔抗ChIFN-γ多克隆抗体进行Western blot分析，表明表达的重组蛋白具有抗原性。     

Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves

Sweetpotato leaves (Ipomoea batatas L.) contain a high content of polyphenolics that consist of caffeic acid, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. We investigated the suppression of the proliferation of selected human cancer cells by phenolic compounds isolated from sweetpotato leaf. The human cancer cells used in this research included a stomach cancer (Kato III), a colon cancer (DLD-1), and a promyelocytic leukemia cell (HL-60). Caffeic acid and di- and tricaffeoylquinic acids dose-dependently depressed cancer cell proliferation, and the difference in sensitivity between caffeoylquinic acid derivatives and each kind of cancer cell was observed. Specifically, 3,4,5-tri-O-caffeoylquinic acid effectively depressed the growth of three kinds of cancer cells, and caffeic acid had an exceptionally higher effect against HL-60 cells than other di- and tricaffeoylquinic acids. In attempting to clarify the mechanism of growth suppression with the addition of the apoptotic inhibitor N-ethylmaleimide, we observed that the nuclear granulation in 3,4,5-tri-O-caffeoylquinic acid-treated HL-60 cells suggested apoptosis induction. This effect was confirmed by DNA fragmentation, an increase of caspase-3 activity, and expression of c-Jun. Growth suppression of HL-60 cells by 3,4,5-tri-O-caffeoylquinic acid was determined to be the result of apoptotic death of the cells. These results indicate that 3,4,5-tri-O-caffeoylquinic acid may have potential for cancer prevention.     

Reproducible induction of cavity spot in carrots and physiological and microbial changes occurring during cavity formation

Inoculation of carrots with 40 types of bacteria, both aerobic and anaerobic, including clostridia isolated from cavity spots, failed to induce cavity spot in carrots. A combined stress of minimum 6 h flooding and temperatures higher than 28°C clearly induced cavity formation. Sugars, amino acids, lipids and minerals leaked from the carrots after flooding and heating the roots. A longer growth period following stress markedly increased cavity spots. Soil types (sandy loam and loess) and several carrot cultivars tested had no marked effect on spot formation. Cavities were formed in stressed carrots grown in sterilized soil containing only one type of bacterium, a Gram-negative short rod. Scanning electron micrographs revealed that after carrots were subjected to combined stress, microscopic cavities nearly free of bacteria were formed under the epidermis. Proliferation of bacteria was concommitant with the appearance of visible cavities. Cell-free extracts of infected carrots showed higher protease and pectinase-specific activities, as well as significantly higher peroxidase and polyphenoloxidase activities and total phenol content as compared to healthy carrots.     

VA菌根真菌对转移Ri T-DNA胡萝卜根器官的侵染

在无菌条件下观察珠状巨孢囊霉（Gigaspora margarita）对转移ＲｉＴ-ＤＮＡ胡萝卜根器官的侵染过程。采用发根土壤杆菌（Agrobacterium rhizogenes）诱导出细胞中整合有ＲｉＴ-ＤＮＡ胡萝卜的不定根作宿主,比较了孢子的４种表面消毒方法,经过２～３个月的双重培养,看到ＶＡ菌根真菌对根器官的侵染,并获得成熟孢子。形成的孢子无须休眠,只要提供发芽条件,就能立即发芽,接种到根的周围又能侵染。文中对侵染的全过程作了较详细的描述,并对此项技术在菌根研究中的意义进行了讨论     

Carrot root size distribution in response to biostimulant application

In 2009–2011, in Poland (53°13′N, 17°51′E), field experiments were conducted concerning the influence of biostimulants Kelpak SL and Asahi SL on the size grades of yield of carrot cv. ‘Karotan’. Kelpak SL is produced from seaweed Ecklonia maxima; Asahi SL is composed of nitrophenols. Biostimulants were sprayed from fourth-leaf stage of carrot, once, twice or three times in growing period. Seaweed extract was used at total doses of: 0, 2, 3, 4, 5, 6 and 7?l?ha^?1, while Asahi SL at 1?l?ha^?1. It was found that biostimulants had a favourable effect on total and commercial root yield, but this effect was dependent on the type of preparation, the dose, the time and the number of treatments. The best results in increasing the commercial yield were achieved after a single application of Kelpak SL at doses 2 or 3?l?ha^?1 at the fourth-leaf stage (13.1% and 12.4% respectively, compared to the control). Both biostimulants Kelpak SL and Asahi SL positively affected the root size distribution by increasing the yield of medium roots (1.9–3.8?cm in diameter) as well as large roots (3.8–5.0?cm), by 30.5% and 15.8%, respectively.     

小麦RPL21基因同源克隆与表达分析

为了解60S核糖体蛋白L21(ribosomal protein L21,RPL21)的基因组结构和表达模式,以GenBank数据库中小麦RPL21基因的部分序列为信息探针,采用RT-PCR和PCR技术从小麦多子房株系幼穗中克隆了RPL21基因的cDNA与DNA片段,并对该基因在小麦多子房近等基因系间的表达差异和多子房...