In situ simultaneous analysis of polyacetylenes, carotenoids and polysaccharides in carrot roots

This paper presents an approach to simultaneously analyze polyacetylenes, carotenoids, and polysaccharides in carrot (Daucus carota L.) roots by means of Raman spectroscopy. The components were measured in situ in the plant tissue without any preliminary sample preparation. The analysis is based on the intensive and characteristic key bands observed in the Raman spectrum of carrot root. The molecular structures of the main carrot polyacetylenes, falcarinol and falcarindiol, are similar, but their Raman spectra exhibit specific differences demonstrated by the shift of their -C[triple bond]C- mode from 2258 to 2252 cm(-)(1), respectively. Carotenoids can be identified by -C=C- stretching vibrations (about 1520 and 1155 cm(-)(1)) of the conjugated system of their polyene chain, whereas the characteristic Raman band at 478 cm(-)(1) indicates the skeletal vibration mode of starch molecule. The other polysaccharide, pectin, can be identified by the characteristic band at 854 cm(-)(1), which is due to the -C-O-C- skeletal mode of alpha-anomer carbohydrates. The Raman mapping technique applied here has revealed detailed information regarding the relative distribution of polyacetylenes, carotenoids, starch, and pectin in the investigated plant tissues. The distribution of these components varies among various carrot cultivars, and especially a significant difference can be seen between cultivated carrot and the wild relative D. carota ssp. maritimus.     

接种丛枝菌根真菌(AMF)对施磷石膏云烟87的生长以及砷污染的影响

【目的】丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)能够促进作物养分的吸收及生长,且对土壤砷污染有一定的抗性。磷石膏(phosphogypsum,PG)因含有丰富的磷、硫等养分可以为作物生长提供必要的养分,同时也可能带来砷污染的风险。【方法】为了探讨接种AMF对云烟87生长的影响以及磷石膏农用可能引起的砷污染风险,通过盆栽模拟试验研究了不同PG添加量(0和40 g/kg以PG0、PG40表示)和接种不同AMF[不接种None mycorrhizal(NM)、接种G.mosseae丛枝菌根真菌(GM)、接种G.aggregatum丛枝菌根真菌(GA)]对云烟87苗期生长及其磷、硫、砷吸收的影响。【结果】试验结果表明:无论接种与否,PG40处理的云烟87植株磷含量、吸收量及吸收效率均显著增加,其地上部硫含量及吸收量也显著增加;除NM处理外,添加PG均显著增加了云烟87根系的硫含量、硫吸收量及吸收效率,并显著增加了其植株的生物量。相同PG添加水平下,与NM处理相比,接种GM显著增加了云烟87根系的磷、硫吸收效率和植株的磷、硫含量及吸收量,另外,GM处理显著降低了其地上部砷含量及吸收量但显著增加了其植株的磷砷吸收比。在PG0处理下,接种GA显著增加了云烟87植株的磷含量及吸收量,并显著增加了其地上部硫含量及吸收量。在PG40处理下,接种GA显著增加了云烟87根系的硫含量和吸收量以及植株的生物量。无论是否添加PG,接种GA不同程度地降低了云烟87地上部砷含量和吸收量从而增加了其地上部的磷砷吸收比。【结论】在所有复合处理中,以添加磷石膏40 g/kg和接种GM对云烟87生长的促进效果较好,对施用磷石膏造成的砷污染有一定程度的抵御作用。     

遗传转化KN1基因促进毛果杨外植体再生

已有研究表明玉米同源异形盒（homeobox）基因Knotted1（KN1）超量表达会导致转基因烟草、拟南芥和番茄等植物中细胞分裂素含量增加,提高转化效率。由于木本植物遗传转化困难,且毛果杨可作为木本植物研究的模式植物,因此,本研究利用农杆菌介导法,将35S启动子驱动的玉米KN1（35S：：KN1）基因导入毛果杨,并分析KN1基因表达对毛果杨再生率的影响。研究结果表明,经GUS组织化学染色及PCR分子鉴定,KN1基因已经成功导入毛果杨幼苗;KN1基因对毛果杨外植体愈伤诱导率影响不明显,但单个外植体芽诱导率比对照高0.96倍,毛果杨的再生频率明显提高;与移栽成活的野生对照植株相比,转化KN1基因植株出现叶片变小,叶色变深,在叶片边缘产生裂片,分枝增加,无顶端优势等特殊表型。本研究中KN1基因引起转化植株表型变化,因此在毛果杨的遗传转化中,可作为一种有效的正向选择标记基因。     

狗蔷薇RaWUS基因组成型表达对烟草叶片形态的影响

为验证从狗蔷薇类原球茎克隆的RaWUS基因的功能,构建了组成型表达载体,并用根瘤农杆菌介导的叶盘转化法对烟草进行了遗传转化。通过除草剂草丁膦筛选,获得了转基因植株并对其进行了表型观察和RT．PCR分析。结果显示转基因烟草叶片形态和叶脉表现出形态变化,包括叶片浅裂、叶片边缘波浪状、叶脉紊乱,甚至叶片的主脉上有不定芽的产生等。RT-PCR分析表明,RaWUS基因仅仅在经过草丁膦筛选的抗性转基因烟草中强烈表达,在野生型烟草中没有表达。表明RaWUS基因已经被成功转入烟草中,并可能通过改变激素的水平和调控维管束的发育来影响叶片形状。     

Impact of egyptian broomrape (Orobanche aegyptiaca (Pers.) parasitism on amino acid composition of carrot (Daucus carota L.)

The relationship between the organic nitrogen status of Egyptian broomrape and one of its hosts, carrot, was studied by comparing amino acid profiles of leaf and root tissues of nonparasitized and broomrape-parasitized carrot plants and by analyzing amino acid profiles of broomrape at different growth stages. Total N concentrations, expressed as a percentage of the dry weight of the tissues, were similar in leaves of nonparasitized and parasitized carrot plants but were lower in parasitized roots than in nonparasitized roots. In both dry and germinated broomrape seeds, N concentrations were lower than or similar to those in broomrape tubercles, shoots, or callus. Individual amino acid concentrations in hydrolysates of leaves of parasitized carrot plants tended to be similar to or greater than those in hydrolysates of nonparasitized carrot plants. Roots of parasitized plants tended to have similar or lower amino acid concentrations than roots of nonparasitized plants. Dry and germinated broomrape seeds had similar amino acid profiles, but individual amino acid concentrations were lower than in the other broomrape tissues examined. The broomrape shoot tended to have lower amino acid concentrations than the tubercle and callus. Free amino acid profiles of leaves and roots of parasitized plants paralleled those of nonparasitized plants, respectively. Individual free amino acids tended to occur at similar or lower levels in dry and germinated broomrape seeds than in the tubercle, shoot, or callus. Free amino acid composition of the broomrape tubercle was similar to that of the parasitized root. Arginine and alanine concentrations in broomrape callus were dramatically higher than those of other amino acids in this or other tissues investigated. These results indicate that changes in the composition of both free and bound amino acids in carrot are associated with broomrape parasitism.     

马铃薯StSnRK1基因过表达提高烟草耐盐性研究

为研究马铃薯蔗糖非发酵-1-型相关蛋白激酶-1基因StSnRK1对于调控植物耐盐性的促进作用,以过表达StSnRK1的烟草株系及野生型为试验材料,研究盐胁迫下StSnRK1对植株生长的影响。耐盐性鉴定结果表明,过表达StSnRK1基因显著提高烟草植株的耐盐性。实时荧光定量PCR(RT-qPCR)分析显示,StSnRK1基因显著上调脯氨酸生物合成相关基因(吡咯琳-5-羧酸合酶NtP5CS)、胚胎发育后期丰富蛋白基因(NtLEA5)和活性氧清除系统相关基因(超氧化物歧化酶NtSOD和过氧化物酶NtPOD)。同时,转基因烟草植株的SOD活性、POD活性和脯氨酸含量显著高于野生型烟草植株,丙二醛(MDA)含量和过氧化氢(H₂O₂)含量显著低于野生型植株。由此可见,StSnRK1基因在改良植物耐盐性方面具有重要作用,为耐盐马铃薯生物技术育种提供了理论基础。     

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.     

优化的VHb基因和融合杀虫基因在烟草中的表达

将人工合成的、密码子优化后的透明颤菌血红蛋白(VHb)基因(vgbM)与含有融合杀虫基因(GFMcryIA和CPTI)表达载体pGBIF4ABC构建成双价基因植物高效表达载体pGBIF4ABCVHB,通过根癌农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum),经PCR及Southernblot检测,证实了双价基因在32株转基因烟草基因组中整合。Westernblot检测证实了vgbM基因的表达,杀虫实验表明,融合杀虫基因表达的毒蛋白具有杀虫活性,转基因烟草平均每株干重高于非转基因烟草植株8%。表明VHb基因和融合杀虫基因烟草在烟草中的表达可使烟草可增产,又具抗虫性。     

优化的VHb基因和融合杀虫基因在烟草中的表达研究

将人工合成的、密码子优化后的透明颤菌血红蛋白（VHb）基因与含有融合杀虫基因（GFMcryIA基因和CPTI基因的融合）表达载体PGBIF4ABC构建成双价基因植物高效表达载体PGBIF4ABCVHB，通过根癌农杆菌介导转化烟草，获得了38株卡那霉素抗性转基因株，经PCR及Southern blotting检测，证实了双价基因在32株转基因烟草基因组中整合。western blot检测证实了VHb基因的表达,杀虫实验表明融合杀虫基因表达出的毒蛋白具有杀虫活性，转基因烟草平均每株干重高于非转基因烟草植株8％。实验结果表明：VHb基因和融合杀虫基因烟草在烟草中的表达既可使烟草既可增产、又具抗虫性。     

毛白杨抗病相关转录因子基因PtWRKY1的表达特性分析

WRKY转录因子作为植物特有的一类重要转录调控因子,广泛参与植物对生物和非生物胁迫的应答反应及形态发育和组织衰老等,已引起人们的关注。本文以前期克隆得到的毛白杨WRKY转录因子基因（命名为PtWRKY1）为研究对象,开展其基因枪介导洋葱表皮细胞瞬时表达、外源信号分子（水杨酸SA,茉莉酸MeJA,脱落酸ABA）诱导表达、转PtWRKY1基因烟草植株的烟草花叶病毒（TMV）接种实验以及抗病相关基因表达的实时荧光定量RT-PCR（qRT-PCR）分析等研究。结果表明,PtWRKY1基因编码蛋白定位于细胞核,SA、ABA及MeJA处理均可诱导PtWRKY1基因表达,但不同诱导因子对PtWRKY1表达量的影响存在一定差异;同时,TMV侵染实验发现外源PtWRKY1表达能增强转基因烟草抗TMV能力,qRT-PCR分析表明PtWRKY1基因及膜保护性酶（POD,SOD,CAT）基因均受TMV诱导而增强表达。本文研究结果可为后续开展毛白杨转录因子PtWRKY1的功能鉴定及应用奠定基础。     

接种丛枝菌根真菌和施用磷石膏对烤烟生长及砷累积的影响

为了探讨丛枝菌根真菌（Arbuscular mycorrhizalfungi,AMF）对烤烟生长及磷石膏农用安全的影响,通过盆栽模拟试验研究了磷石膏（Phosphogypsum,PG）不同添加量[0、40mg·g^-1（PG0、PG40）1和接种两种AMF[Glomus massecle（GM）、G．aggregatum（GA）】对烤烟（KRK26）苗期生长及其磷（P）、硫（S）、砷（As）吸收的影响。试验结果表明：无论是否接种AMF,磷石膏的添加均显著增加了KRK26地上部生物量及其植株s含量、吸收量及吸收效率;除不接种处理（NM）的烤烟根系外,PG40处理显著增加了KRK26植株P含量、吸收量及吸收效率,并显著降低了NM处理的地上部As含量及吸收量,进而增加了磷砷吸收比。相同PG添加水平下,与不接种相比,接种GM和GA均显著增加了KRK26植株的生物量。除PGO处理的烤烟根系外,接种GM显著增加了KRK26植株P、S含量与吸收量及吸收效率,以及植株As含量及吸收量,并显著增加了PG40处理的植株磷砷吸收比;接种GA也显著增加了KRK26植株P、S含量及吸收量,并显著降低了PG0处理地上部As含量及吸收量。所有复合处理,以添加磷石膏40mg·g^-1和接种GA处理对KRK26的生长促进效果较好,对磷石膏施用造成的As污染有一定抵御作用。     

应用激光拉曼光谱判别油菜叶片核盘菌早期侵染

病原物核盘菌侵染油菜植株所引发的油菜菌核病严重制约着油菜产业的发展,及早诊断核盘菌的侵染有助于油菜菌核病的早期防治。病原物一般由侵入点向植物寄主四周扩散形成病斑,而与病原物侵染点不同距离的组织区域可代表病害的不同严重程度。该研究采用激光共聚焦显微拉曼光谱仪在800~2 000 cm-1范围内获取健康和染病油菜叶片的拉曼光谱曲线,接着采用小波变换(wavelet transform,WT)进行拉曼光谱的预处理以去除荧光背景的干扰,然后选择主成分因子(principal components,PC-1和PC-2)以及特征参量(1 006,1 156和1 522 cm-1)进行样本间的聚类分析,最后分别基于主成分因子和拉曼特征参量建立最小二乘支持向量机(least squares support vector machine,LS-SVM)进行菌核病侵染油菜叶片不同阶段的判别分析。结果发现采用基于PC-1主成分,1 156和1 522 cm-1处的拉曼强度建立的LS-SVM判别模型可以得到100%的识别率。研究结果表明,通过判别分析油菜叶片菌核病病斑不同区域处的拉曼光谱可以实现核盘菌侵染油菜叶片的早期判别,这为后续探究植物病害的早期监测以及研发油菜叶片菌核病早期诊断拉曼便携仪提供理论参考。     

Compartive study of volatile components and fatty acids of plants and in vitro cultures of parsley (Petroselinum crispum (Mill) nym ex hill).

Volatile compounds from plants, callus tissue cultures, and cell suspensions of parsley (Petroselinum crispum) were captured during the growth cycle using a dynamic headspace extraction and were identified by gas chromatography-mass spectrometry. Parsley plants were found to produce mainly monoterpenes, and the compound of major abundance was p-1,3,8-menthatriene, followed by beta-phellandrene and apiole. Callus cultures and cell suspensions produced aldehydes (nonanal and decanal) that were also detected in parsley plant. The former also produced limonene, acetophenone, and benzotiazol; these were not observed in the plants. The production of volatiles in plants, callus tissue, and cell suspensions was found to be time-dependent. Free and bound fatty acids were also monitored by an in situ method. Palmitic (C16:0) and stearic (C18:0) acids were the most abundant fatty acids in all materials; however, higher levels were found in plants. On the other hand, the unsaturated C16:1 and C16:3 were not detected in the in vitro cultures.     

Resistance of soybean vegetative storage proteins (S-VSPs) to proteolysis by rumen microorganisms

Soybean vegetative storage proteins (S-VSPs) are lysine-rich and, hence, are potentially of high nutritive value for high productive ruminants. Using S-VSPs from wild-type soybean and from transgenic tobacco plants expressing either one of the two S-VSPs subunits (S-VSP alpha or S-VSP beta) or both, we tested their stability in cow rumen fluid under in situ conditions, using SDS-polyacrylamide gel electrophoresis. Proteolysis and degradation pattern of S-VSPs from transgenic tobacco leaves occurred relatively fast compared with that of wild-type (WT) soybean plants. Comparing the two S-VSPs subunits expressed in transgenic plants, we found that S-VSP alpha was degraded much faster than S-VSP beta. The degradation pattern of S-VSPs in transgenic tobacco plants expressing both subunits resembled that of WT soybean. In contrast, the degradation pattern of transgenic tobacco plants expressing a single subunit was different. These finding suggest that the quaternary structure of S-VSPs may be an important factor determining their resistance to rumen degradation. Our results also suggest that the stability to rumen proteolysis of a given protein, when expressed in a transgenic plant, may not always be predictable and has to be verified.     

大熊猫生长激素基因Am-GH的表达载体构建及其表达研究

植物反应器生产药品成本低,具有改变传统制药方法的光明前景。本研究 以PCR法克隆的大熊猫生长激素基因Am-GH,构建了原核表达载体pEGX-AmGH和植物表达载体pCAMBIA13011-GH。pEGX-AmGH转化E. coli得到高效表达。pCAMBIA13011-GH通过根癌农杆菌GV3101的介导转化烟草无菌叶片外植体,经过四轮潮霉素(Hygromycin, Hyg)递增筛选,获得抗性植株。其中部分Hyg抗性植株经GUS和PCR检测为阳性。鉴定结果表明Am-GH 已成功整合到烟草基因组中。为进一步探讨大熊猫生长激素基因Am-GH在烟草中的表达奠定了基础。     

用γ射线和叠氮化钠诱变的玉米愈伤组织筛选耐旱和雄性不育材料

用60Coγ射线和叠氮化钠(NaN3)处理玉米愈伤组织,在含1.0%NaCl的高渗培养基上筛选,对再生植株株系进行耐旱性鉴定。结果表明,20 Gy的辐射和1 mmol/L NaN3是较为适合玉米愈伤组织诱变的处理组配。22个再生植株株系中,有5个诱变株系的耐旱性高于未诱变对照,其中1个株系的耐旱性与耐旱自交系“81565”接近。此外,从M1代株系中发现了1个雄性不育株,鉴定为细胞核隐性单基因控制的孢子体雄性不育。     

冷诱导基因转录因子CBF1的组成型表达对植物的抗寒性及生长发育的影响

通过农杆菌介导法将玉米泛素启动子(Pubi)调控的CBF1基因以及CaMV35启动子调控的GUS基因一起转化烟草,研究了冷诱导基因的转录因子CBF1(CRT/DRE binding factor 1)对植物抗寒性及生长发育的影响。结果表明:CBF1的组成型表达增强了冷敏感植物的抗寒性;在表型上使植株矮壮,叶片着生密集,叶色深绿,叶厚度增加;促进侧枝生长,延长营养生长期,推迟花期;对植株的结实性无明显影响。植株抗寒能力以及表型变化程度与CBF1的表达强度相关。因此,利用CBF1基因进行植物遗传改良时应根据物种的应用特点采用合适的表达调控策略。     

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.     

芹菜水孔蛋白基因AgPIP2;1的克隆及其对非生物胁迫的响应

为了研究芹菜质膜内在蛋白基因的序列特征及其在非生物胁迫条件下的表达特性,探讨其抗逆功能,以芹菜品种六合黄心芹为试验材料,通过RT-PCR技术克隆AgPIP2;1基因,通过生物信息学软件分析其核苷酸和氨基酸序列特征,并采用荧光定量PCR技术检测其在不同组织的表达及其对非生物胁迫的响应。结果表明,AgPIP2;1基因含有1个861 bp的开放阅读框,编码286个氨基酸,其编码的蛋白属于PIP2类蛋白。氨基酸序列分析显示,AgPIP2;1含有高度保守的NPA基序以及高等植物PIP蛋白的保守序列,与其他物种PIP2类蛋白具有较高的同源性,与胡萝卜DcPIP2;1的氨基酸序列同源性达到97.90%。在亲缘关系上与大豆GmPIP2;1、胡萝卜DcPIP2;1和绿豆VrPIP2;1较为接近。实时荧光定量PCR技术分析表明,AgPIP2;1基因在芹菜根、叶柄和叶片中均有表达,其中在叶片中的表达量最高,在根中的表达量最低,呈现比较明显的组织特异性。此外,AgPIP2;1基因受到高温、低温、干旱和盐等非生物胁迫的诱导,说明该基因可能参与芹菜抵御非生物胁迫的过程。本研究为进一步了解AgPIP2;1基因的功能及其在非生物胁迫中的作用奠定了基础。     

玉米花青素调节基因Lc对转基因烟草和矮牵牛花色的影响

利用PCR方法从油菜(Brassica napus)基因组中扩增得到花瓣特异性启动子XY355,构建该启动子与玉米(Zea mays)花青素调节基因Lc的植物表达载体pXY60。通过根癌农杆菌(Agrobacterium tumefaciens)介导,将pXY60分别转化烟草(Nicotiana tabacum)和矮牵牛(Petunia hybrida)。对再生植株进行PCR检测,结果表明,外源基因已经整合到转基因烟草和矮牵牛的基因组中。观察发现,转基因植株的表型发生了明显变化:转基因烟草的花色由浅红变成了红色,转基因矮牵牛的花色由白色变成了浅紫色。