Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot (Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis （Southern and Western） of callus tissue （non-photosynthetic tissue） to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.

原生质体非对称融合获得胡萝卜(Daucus carota L.)种内胞质杂种

用紫外线辐射处理胡萝卜雄蕊瓣化型不育材料 7 0 8(供体 )的原生质体 ,与经 15mmol/L碘乙酰胺预处理的来源于可育材料 6 6 3的原生质体 (受体 )电融合 ,获得了 33株再生植株。所有再生植株营养体形态特征与受体亲本一致 ,染色体数目为 18条 ,是二倍体。RAPD分析表明 ,所有再生植株核基因型与受体亲本一致 ;线粒体特异性引物 -STS4 引物PCR扩增中 ,所有再生植株均得到了与供体亲本一致的谱带 ,而与受体亲本不同 ,认为 33株再生植株均为胞质杂种。对 4株再生植株进行花期形态学鉴定 ,全部为雄蕊瓣化型不育株 ,进一步证实获得的为胞质杂种 ,雄蕊瓣化型细胞质不育性已由 7 0 8供体转移到受体可育材料 6 6 3中。

胡萝卜Daucus carotavar.sativus Hoffm Deutschl抗冻蛋白基因的克隆及测序

以宁夏吴忠胡萝卜、陕西华县胡萝卜和陕西汉中胡萝卜 3个地方品种为材料 ,用PCR (polymerasechainreaction)的方法克隆了中国胡萝卜的抗冻蛋白基因 (afp) ,测定了其核苷酸序列 ,并和英国胡萝卜的afp序列进行了对比。在所测 10 0 4个核苷酸中 ,两变种碱基不同者有 36个 ,占 3.6 % ,其中无义突变 2 1个 ,有义突变 15个。按有义突变计 ,同源性为98.5%。

胡萝卜(Daucus carota L.)胚性细胞蛋白的分离研究

应用IEF/SDS-PAGE双向电泳技术,比较了胡萝卜胚性细胞、非胚性细胞和不同发育时期的胚状体中可溶性蛋白的双向电泳图谱,结果发现在胚性细胞中特异存在的胚性细胞蛋白在不同发育时期的胚状体中也存在,但在非胚性细胞中不存在。因此,推测体细胞胚胎发生所需的一些基因在胚性细胞中就早已表达了。我们还成功地分离和测定了ECP 45-2 N-末端和中央部分氨基酸序列。与已知氨基酸序列相比,ECP 45-2部分氨基酸序列与ECP 45-1 部分氨基酸序列具有较高比例的同源性。因此, ECP 45-1和45-2可能属于同一因基家族。

NAA对胡萝卜(Daucus carota)悬浮细胞茄红素合成的影响

通过对胡萝卜新透心红 DaucuscarotaCV.XinTouxinhong 细胞悬浮系单位鲜重细胞的茄红素含量、细胞总产量以及茄红素总产量的分析比较,研究了培养基中不同NAA浓度对胡萝卜细胞悬浮系茄红素代谢的影响.结果表明,NAA具有增强细胞茄红素代谢水平、提高单位鲜重细胞中茄红素的含量的作用,但对细胞的生长具有抑制作用.从茄红素单产和总产量综合评价,培养基中加入3.0mg·L-1NAA效果最佳.

细胞转化获得转白细胞介素-4基因胡萝卜(Daucus carrot)

本研究以胡萝卜‘开拓者’无菌苗胚轴诱导的愈伤组织经振荡培养获得悬浮培养细胞为受体材料,以卡那霉素抗性基因为选择标记基因,对农杆菌介导的胡萝卜细胞遗传转化体系进行了优化。结果表明：悬浮细胞预培养1-3d,胡萝卜细胞与农杆菌细胞比例为1：10或1：100,共培养时间为1-3d时,转化效率较高;但以悬浮细胞预培养1d,胡萝卜细胞与农杆菌细胞比例为1：10,共培养时间为1d时,可得到较多的转化苗。共获得抗性苗290株,经PCR检测66株扩增出特异性条带;部分阳性植株经PCR-Southern检测,初步证明目的基因已转入到胡萝卜中,并收获了转基因胡萝卜。

锰胁迫对野胡萝卜(Daucus carota)幼苗生长及理化特性的影响

野胡萝卜(Daucus carota)具有生物量大、适应性强、株型美观等特点,是锰矿废弃地污染治理的重要备选植物。以野胡萝卜为试材,采用盆栽试验的方法,研究了不同锰浓度(0～2 000 mg·kg^(-1))条件下野胡萝卜幼苗的生长及理化指标的变化特征。结果表明:随着锰浓度的升高,野胡萝卜幼苗生长呈现出‘低促高抑’现象,100～500 mg·kg^(-1)锰胁迫可有效促进植物幼苗生长;当锰浓度高于1 500 mg·kg^(-1)时,幼苗的生长受到抑制,其株高、根长、地下部干质量均较对照降低。随着锰浓度的升高,野胡萝卜幼苗叶片叶绿素、可溶性糖、可溶性蛋白质、游离脯氨酸含量以及超氧化物歧化酶(SOD)和过氧化物酶(POD)活性均呈现先增加后下降的变化趋势,并在500 mg·kg^(-1)锰胁迫下达到最大值;而过氧化氢酶(CAT)活性则表现为逐渐下降趋势,丙二醛(MDA)含量则表现为先下降后上升的趋势。研究表明,野胡萝卜可通过增加渗透调节物质浓度和增强抗氧化酶活性等方式应对锰胁迫,从而在较高浓度锰胁迫下维持植株正常生长,表现出较强的耐锰胁迫能力。

In vivo antioxidant and hepatoprotective activity of methanolic extracts of Daucus carota seeds in experimental animals

Objective:To assess the In vivo anlioxid Fanl and hepaloproleclive activity of metlianolic exlracl of Daucus carota(D.carota) seeds in experimental animals.Methods:Methanolic extracts of D.carota seeds is used for hepatoproleclion assessment.Oxidative stress were induced in rats by thioacetamide 100 nig/kg s.c.in four groups of rats(two test,standard and toxic control). Two test groups received D.carota seeds extract[DCSE) at doses of 200 mg/kg and 400 mg/kg. Standard group received silymarin(25 mg/kg) and toxic control received only thioacetamide. Control group received only vehicle.On the 8th day animals were sacrificed and liver enzyme like serum glutamic pyruvic transaminase(SGPT),serum glutamic-oxaloacetic transaminase(SCOT) and alkaline phosphatase(ALP)were estimated in blood serum and antioxidant enzyme like superoxide disnuituse(SOD),cululase(CAT),glutathione reductase(CKD),glutathione peroxidase(GPX),glutalhione-S-transferase(GST)and lipid peroxidation(LPO)were estimated in liver homogcnatc.Results:A significant decrease in SGPT,SCOT and ALP levels was observed in all drug treated groups as compared to thioacetamide group(P<0.001) and in case of antioxidant enzyme a significant(P<0.001) increase in SOD.CAT,GRD,GPX and GST was observed in all dmg treated groups as compared with thioacetamide group.But in case of LPO a significant(P <0.001) reduction was observed as compared to toxic control group.Conclusions:DCSE has contributed lo the reduction of oxidative stress and the protection of liver in experimental rals.

Components of Essential Oils in Different Parts of Daucus carota L. var. sativa Hoffm

The components of the essential oils obtained from different parts of Daacus carota L.var. saliva Hoffm were analyzed. The percentages of the essential oils extracted are 0. 27% （ mL/100 g material） for the flowers, 0. 0/% for the stems and leaves and 0. 01% for the roots. Fifty-four, Sixty-six and Thirty-three compounds were,respectively, separated and identified from the flowers, the stems and leaves and the roots, among which unsaturated alkene compounds are thirty-nine, thirty-nine and twenty-one, respectively, accounting in turn for up to 90. 21%, 90. 49% and 72. 65% of the total essential oils. Because alkene compounds have double bonds that are easily oxidized, it can he inferred that the components of the essential oils in the different parts of Daucus carota L. var. saliva Hoffm should show an activity of the antioformation of free radicals to some extent.

Asymmetric Reduction of Heteroaryl Methyl Ketones Using <i>Daucus carota</i>

Asymmetric reduction of the heteroaryl prochiral ketones to corresponding chiral alcohols by Daucus carota was studied. The study highlights selective bioreduction of different substituted heteroaryl ketones (1a - 1j) to their respective chiral alcohols (2a - 2j) using plant dehydrogenase enzymes present in Daucus carota in good yields (60% - 95%) and enantioselectivity (76% - 99%) with S-form configuration. The results obtained confirm that the membrane bound dehydrogenase enzyme has broad substrate specificity and selectivity in catalyzing both six and five membered heteroaryl methyl ketones. The present methodology demonstrates promising and alternative green route in the synthesis secondary chiral alcohols of biologically importance in a simple, inexpensive and eco-friendly process.

Dc GA20ox2 and DcGA2ox1 alter endogenous gibberellin contents and adjust lignin accumulation in carrot

Gibberellins(GAs)are a class of plant hormones that can affect plant growth and development.GA-oxidases are rate-limiting enzymes,which play a direct role in GA accumulation in plants.However,the roles of GA-oxidase on carrot(Daucus carota L.)taproot development are still unclear.In this study,two GA-oxidase genes,DcGA20ox2 and DcGA2ox1,were identified in carrot.Transgenic carrot plants were obtained by using Agrobacterium-mediated genetic transformation method.The results showed that overexpression of DcGA20ox2 significantly promoted the accumulation of active GAs in carrot,increased plant height,generated more branches,and enhanced xylem development.Overexpression of DcGA2ox1 significantly reduced the total contents of active GAs compared with the control group,resulting in a dwarf phenotype and markedly increased lignin content of the transgenic carrot.The expression profiling showed that the genes of GA metabolic pathway responded to the negative feedback regulation mechanism.At the same time,the expression of most genes in lignin biosynthesis and polymerization process was up-regulated,corresponding to the massive accumulation of lignin.These findings indicated that DcGA20ox2 and DcGA2ox1affected carrot growth and development by regulating the levels of endogenous GAs.The results from current work might shed light on further studies aimed to regulate lignification in carrot and other crops.

Synthesis of betanin by expression of the core betalain biosynthetic pathway in carrot

Betalain has received increased attention because of its high nutritional value and crucial physiological functions.Based on the elucidation of its core biosynthetic pathway,betalain can be produced in additional plants by metabolic engineering.Synthesis of betalain in carrot(Daucus carota L.)can improve its nutritional quality and economic value by extracting betalain from the fleshy root,non-edible part,and processing residue of carrot.In this study,two different constructs,namely,pYB:mCD(AomelOS,BvCYP76AD1S,and BvDODA1S)and p YB:CDD(BvCYP76AD1S,BvDODA1S,and MjcDOPA5GTS),were introduced into carrot for betanin synthesis by Agrobacterium-mediated transformation.Betanin can be synthetized in both transgenic calli,and p YB:m CD-transgenic callus can be used to produce betacyanin by suspension culture.However,pYB:mCD-transgenic seedlings can synthetize betanin only by tyrosine feeding.The p YB:CDD-transgenic lines can synthetize betanin in whole plants.The betanin content in fleshy root of pYB:CDD-transgenic carrot was(63.4±9)μg·g^(-1)fresh weight according to quantitative analysis.These betanin-producing carrot plant materials can be used to synthesize betanin for industrial application or consumption as dietary sources.

Isolation of development-related genes in somatic embryo radicle of carrot (Daucus carota L.) using modified cDNA RDA

Using modified cDNA RDA capitalizing on the high affinity of streptavidin for biotin and magnetic-absorption-based separation, we have obtained four bands of specifically expressed cDNA in the carrot somatic embryo deregulated for 12 h, which were designated as NR-1, NR-2, NR-3 and NR-4, respectively. As revealed by homology analysis of their DNA sequences after cloning them into pBS, remarkable homology was demonstrated in NR-2, NR-3 and NR-4 with the genes coding for LEA (late embryogenesis abundant protein), Dna J and xyloglucan endo-trans-glycosylase in plants. On the other hand, NR-1 showing no homology with any known sequence may have come from unknown genes. Using 32P-labeled NR-1 as probe, hybridization with cDNA fragment population has shown that we have actually cloned a new gene fragment related to radicle development. As shown by further Southern hybridization, these genes may be present in carrot genome in the form of single or low copies.

Nutritional and Health Benefits of Carrots and Their Seed Extracts

Carrot is a root vegetable with carotenoids, flavonoids, polyacetylenes, vitamins, and minerals, all of which possess numerous nutritional and health benefits. Besides lending truth to the old adage that carrots are good for eyes, carotenoids, polyphenols and vitamins present in carrot act as antioxidants, anticarcinogens, and immunoenhancers. Anti-diabetic, cholesterol and cardiovascular disease lowering, anti-hypertensive, hepatoprotective, renoprotective, and wound healing benefits of carrot have also been reported. The cardio- and hepatoprotective, anti-bacterial, anti-fungal, anti-inflammatory, and analgesic effects of carrot seed extracts are also noteworthy. All are discussed in this review article.

Metabolic Engineering of Carotenoid Formation in Carrot Roots

Carotenoids, a large class of yellow, orange and red pigments accumulate in the chloroplasts of leaves or in the chromoplasts of many flowers and fruits as well as carrot roots. In plants, carotenoids function as

胡萝卜(Daucus carota var. sativa)密码子偏好性及属内质体基因组核苷酸多样性分析

核苷酸多样性作为衡量物种遗传变异水平的重要指标,对于研究物种遗传多态性的高低、进化历史和系统位置具有重要的科学意义,密码子偏好性则跟基因表达和自然选择压力等密切相关。本研究通过计算胡萝卜属(Daucus L.) 17种植物的叶绿体基因组片段的核苷酸多样性,开发出了可用于系统学和群体遗传学等研究的叶绿体基因分子标记;并且基于Codon W和CUSP统计数据,通过相关性分析、中性绘图分析、ENC-plot分析以及PR2-plot分析,探究了胡萝卜(Daucus carota var. sativa)密码子使用偏好性。结果表明,有效密码子数(ENC)与最优密码子使用频率(FOP)、密码子适应指数(CAI)、密码子偏好指数(CBI)具有显著的相关性,胡萝卜叶绿体基因的密码子偏好性主要受到突变和自然选择的作用,其中,突变的影响更大。此外,本研究确定了5个高表达基因和18个最优密码子。这些研究结果有助于探究胡萝卜的进化历史和基因的表达,从而为品种改良和提高产量提供理论支持。

基于广靶代谢组学分析胡萝卜萜类代谢产物差异

【目的】萜类代谢物是评价胡萝卜风味品质的主要指标,对胡萝卜口感风味品质的形成具有重要作用。通过比较甜味浓烈组(-3)和苦味浓烈组(3)两组不同品种胡萝卜成熟期肉质根风味成分的代谢谱,为胡萝卜风味特征成分鉴定及优异资源的开发利用提供参考。【方法】建立高效的、具有高灵敏度和高通量的代谢组学分析平台和方法:采用UPLC-MS/MS方法对胡萝卜萜类代谢物进行分析测定,鉴定影响胡萝卜风味品质的萜类代谢物。采用OPLS-DA结果的VIP值、log2FC值筛选差异性代谢物(设置阈值:VIP>1、fold change≥2和fold change≤0.5)。【结果】利用UPLC-MS/MS技术对胡萝卜成熟期肉质根进行广泛靶向代谢组学测定,共筛选出50种显著差异的萜类代谢物,12个上调,2个下调。其中,显著上调的分别是京尼平(0.754)、粳米素B(0.936)、氧叶烯A(1.03)、乙酸香芹酯(1.04)、3-(3'-羟基丁基)-2,4,4-三甲基环己烷-2,5-二烯酮(0.662);显著下调的是脱氢地芰普内酯(0.617)。从含量来看,苦味胡萝卜中萜类代谢物含量综合较高的分别是甲黑素甲苷A(16.98)、莪术醇(17.57)、京尼平(15.32),甜味胡萝卜中萜类代谢物含量综合较高的分别是甲黑素甲苷A(17.61)、异香附醇(16.01)、莪术醇(18.73)。根据P值来看,排名前5的分别是薇甘菊内酯(0.994)、寥二醛(0.991)、甲基(E)-5,11-二羟-4-((Z)-2-甲基丁-2-烯酰氧基)-3,10-二亚甲基-2-氧代-2,3,3a,4,5,8,9,10,11,11a-十氢环癸并[b]呋喃-6-羧酸酯(0.978)、(3R)-3-羟基-紫罗兰酮(0.974)、布卢门醇A(0.969)。采用聚类分析方法对两组胡萝卜肉质根中的差异代谢物进行分析,其中,分组3中的代谢物含量要明显高于分组-3中的代谢物含量。利用KEGG进行差异代谢物注释和富集分析。【结论】萜类代谢物是影响胡萝卜风味的主要化合物,其中,京尼平、粳米素B、氧叶烯A、乙酸香芹酯、甲黑素甲苷A、莪术醇、异香附醇、脱氢地芰普内酯、薇甘菊内酯等萜类代谢物可能是影响胡萝卜风味的关键成分。

南鹤虱水提物及乙醇提取物对小鼠皮肤刺激性的初步研究

目的:研究南鹤虱的水提物和乙醇提取物对皮肤产生刺激性反应,以评价其安全性。方法:参考国家食品药品监督管理总局发布的《中华人民共和国国家标准化妆品安全性评价程序和方法》,通过皮肤刺激性实验,分析南鹤虱的水提物及乙醇提取物皮肤应用的安全性,为南鹤虱的开发利用提供理论依据。结果:一次给药皮肤刺激性实验中,小鼠的皮肤反应刺激强度得分均小于0.5分;连续多次给药皮肤刺激性实验中,小鼠的皮肤反应刺激强度得分均为0分,表明南鹤虱的水提物和乙醇提取物对小鼠的正常皮肤及划伤皮肤均无刺激性。结论:南鹤虱的水提物和乙醇提取物对小鼠的正常皮肤及划伤皮肤均无刺激性,为今后开发南鹤虱护肤的相关产品提供一定的参考价值。

胡萝卜抗冻蛋白在大肠杆菌中的高效表达

以pET - 11a为载体构建了胡萝卜抗冻蛋白非融合表达的重组质粒 ,并在大肠杆菌菌株BL2 1(DE3,pLysS)中成功地进行了高效表达。表达的蛋白质以包涵体的形式存在 ,相对分子质量为 36 80 0。经正交试验得到了优化的诱导表达条件。包涵体经各种缓冲液洗涤纯化、溶解、复性和超滤浓缩后 ,经SDS -PAGE电泳检测 ,纯度达单一带水平。生物学活性研究表明 ,重组表达的胡萝卜抗冻蛋白具有很强的热滞活性和提高细菌耐寒性的作用。

微波提取对胡萝卜中的多糖基本结构特征及固体形貌的影响

研究微波提取对胡萝卜中的多糖基本结构特征及固体形貌的影响。采用沸水浴、微波两种方式提取胡萝卜中的多糖(分别记为WEP和MEP),通过基本理化性质测定、高效体积排阻色谱(high performance size exclusion chromatography,HPSEC)和单糖组成分析等方法比较了两者的基本结构特征,并采用扫描电子显微镜对两者的固体形貌进行表征。结果显示,WEP和MEP的中性糖、糖醛酸和总酚含量存在差异,两者均由半乳糖醛酸、半乳糖、葡萄糖、阿拉伯糖、鼠李糖组成,但MEP总糖含量更高并含有更多果胶类单糖;WEP和MEP的HPSEC图有一定相似性,但WEP中含有重均分子质量高达7.77×105 D的组分;WEP表现较为聚集的片状结构,而MEP组分则表现较为分散且主要为小片段状态。上述结果表明,两种不同提取方法所得到的多糖具有显著差异。