Two new eudesmane sesquiterpenes from Lactuca sativa var.anagustata L

Two new eudesmane sesquiterpene lactones were isolated from the stalk of Lactuca sativa vat anagustata L and their structures were elucidated by means of spectroscopic methods, including 2D NMR （1H-1H COSY, HMBC and NOESY） as 1β-O-β-D- glucopyranosyl-4α-hydroxyl-5α, 6β, 11βH-eudesma-12, 6α-olide （1） and 1β-hydroxyl-15-O-（p-methoxyphenylacetyl）-5α, 6β, 11 βH-eudesma-3-en- 12, 6a-olide （2）.

TDZ对莴苣(Lactuca sativa L.)器官发生及乙烯形成的影响

为了研究TDZ在植物器官发生中的生理作用，用不同浓度的TDZ替代MS中的BA，发现仅用1／50BA浓度（0．1mg／L）的TDZ处理，已相当于用BA处理所得芽数，可见它具有很强的细胞分裂素活性；但是TDZ又能显著促进乙烯形成，较低浓度时抑制莴苣的器官发生，可能此时乙烯起着主导作用。

Selenium distribution and nitrate metabolism in hydroponic lettuce(Lactuca sativa L.): Effects of selenium forms and light spectra

A deficiency in selenium(Se) in the human diet is a worldwide problem. The intake of Se-rich vegetables can be a safe way to combat Se deficiency for humans. However, most leafy vegetables can accumulate a high content of nitrates, which poses a potential threat to human health. Light is an important environmental factor that regulates the uptake and distribution of mineral elements and nitrogen metabolism in plants. However, the effects of Se forms and light conditions, especially light spectra, on the uptake and translocation of Se and on nitrate reduction are poorly understood. In this study, lettuce(Lactuca sativa L.) was treated with exogenous Se applied as selenate(10 mmol L^-1) and selenite(0.5 mmol L^-1) and grown under five different light spectra: fluorescent light(FL), monochromatic red LED light(R), monochromatic blue LED light(B), and mixed red and blue LED light with a red to blue light ratio at 4(R/B=4), 8(R/B=8), and 12(R/B=12), respectively. The effects of light spectra and Se forms on plant growth, photosynthetic performance, Se accumulation and nitrate reduction were investigated. The results showed that the light spectra and Se forms had significant interactions for plant growth, foliar Se accumulation and nitrate reduction. The Se concentration and nitrate content in the leaves were negatively correlated with the percentage of red light from the light sources. Compared to Se applied as selenite, exogenous Se applied as selenate was more effective in reducing nitrate via promoting nitrate reductase and glutamate synthase activities. The lowest nitrate content and highest plant biomass were observed under R/B=8 for both the selenate and selenite treatments. The significant effect of the light spectra on the root concentration factor and translocation factor of Se resulted in marked variations in the Se concentrations in the roots and leaves. Compared with FL, red and blue LED light led to significant decreases in the foliar Se concentration. The results from this study suggest that the light spectra can contribute to Se distribution and accumulation to produce vegetables with better food quality.

Effects of Priming in Combination with Fungicides on Germination and Infestation of Lettuce (Lactuca sativa L.)

Two seed lots of lettuce (Lactuca sativa L.) cv. Krlowa Majowych were primed in polyethylene glycol (PEG) 8000 at -1.25MPa for 7 days in dark at 20C. Two fungicide treatments combined with PEG were used to control fungi: 1) Funaben T(0.1%) and Apron 35 SD (0.02%) were added to -1.25 MPa PEG solution (PEG+F); 2) After seed priming and drying,Funaben T was mixed at 5 g kg-1 and Apron 35 SD at 1 g kg-1 of seeds (PEG then F dust). PEG, PEG+F and PEG then F dusttreatments reduced significantly thermodormancy and increased the seed germination at 35C in both lots. PEG then Fdust treatment had the best effect on reducing seed thermodormancy at 35C, and increased seed germination to 64.5% inlot 1 and 72.0% in lot 2 compared to 0 in untreated. PEG and PEG then F dust treatments have accelerated germinationspeed at 20 and 15C in both lots. There was a significant reduction in seed infestation in which the seeds were treatedwith PEG+F and PEG then F dust compared to untreated seeds and PEG primed seeds. PEG priming enhanced seedinfestation considerably by Alternaria alternata, Penicillium spp., Rhizopus spp. and Ulocladium spp. Mycologicalanalysis showed higher frequency of Alternaria alternata and Ulocladium spp. in untreated seeds at 20C. Priming incombination with fungicides significantly alleviated the infestation of fungi, especially Alternaria alternata infestation.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

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.

Construction of Lactuca sativa Plastid Transformation Vector and High-level Expression of gfp Gene in Escherichia coli

Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.

Effects of Different Concentrations of Photosynthetic Bacteria Solutions on Yield and Quality of Lactuca sativa L.

The effects of different concentrations of photosynthetic bacteria solutions （CK, 0 ml photosynthetic bacteria agent＋4 000 ml nutrient solution; T1, 200 ml pho- tosynthetic bacteria agent＋3 800 ml nutrient solution; T2, 400 ml photosynthetic bacteria agent＋3 600 ml nutrient solution; T3, 600 ml photosynthetic bacteria agent＋ 3 400 ml nutrient solution; T4, 800 ml photosynthetic bacteria agent＋3 200 ml nutrient solution; and T5, 1 000 ml photosynthetic bacteria agent＋3 000 ml nutrient solu- tion） on the leaf number, fresh mass, root vigor, vitamin C content, soluble sugar content, soluble protein content and nitrate content of hydroponic Lactuca sativa L. were investigated. The results showed that the leaf number was increased by 62.66%, the fresh mass was increased by 139.7%, the root vigor was increased by 132.04%, the vitamin C content was increased by 18.34%, the soluble protein content was increased by 16.60%, the soluble sugar content was increased by 192.37%, and the nitrate content was reduced by 69.44% in the T3 group com- pared with those in the control group. The photosynthetic bacteria solution in the T3 group reduced the content of nitrate and improved the yield and quality of hydroponic L. sativa.

Environmantal Risk Phytoaccumulation of Arsenic in Spinacea Olaracea L, and Lactuca Sativa L

Pollution of the environment, particularly the soil, appears to be one of the major contemporary issues. In addition, many studies point to the involvement of ecological risk of heavy metals into the food chain through edible plants like Spinaceae oleraceae L. and Lactuca sativa L.. This paper presents the results of the ＂ex-situ＂ application of phytoacumulation on the soils of contaminated terrain using the plant species of spinach （Spinacea oleracea L.） and lettuce （Lactuca sativa L.）. Sequestration of arsen from the soil by these plant species was observed through phytoaccumulation factor--PF and limit values （mg/kg）. The main task and objective of this research was to establish the level of accumulation of toxic element As from the soil by plants and to calculate the PF factor of transfer. The experiment was set up in the control conditions where the soil from eight contaminated locations was placed in the experimental containers. The AAS method was used to analyze heavy metal in plant material and soil. PF factor values ranged from 0.1 to 1.0 which indicates that the spinach and lettuce plants rechargeable moderate compared to arsenic.

叶用莴苣热激蛋白基因LsHsp70-2711的克隆及高温胁迫下的功能分析

【目的】通过克隆Hsp70相关基因,并利用VIGS分析叶用莴苣热胁迫下Hsp70表达量和形态变化,为解析热激蛋白基因Hsp70在高温胁迫下的响应机制及分子机理奠定基础。【方法】通过同源克隆及RACE技术,获得叶用莴苣热激蛋白LsHsp70-2711的cDNA全长序列,利用实时荧光定量PCR(qRT-PCR)分析该基因在不同温度和不同高温时间下的叶用莴苣热敏品种‘P-S11’和耐热品种‘G-S59’的表达差异,确定基因和高温的相关性。根据VIGS技术,构建p TRV-LsHsp702711瞬时沉默载体,转化农杆菌GV1301,注射法侵染叶用莴苣叶片,三周后经PCR鉴定得到阳性植株。对照组和阳性组在基因表达和形态上进行对比,对照植株和阳性植株热胁迫和干旱处理后再次分析LsHsp70-2711的表达特性,观测形态变化。【结果】LsHsp70-2711 cDNA全长为2 226 bp,开放阅读框为2 154 bp,编码718个氨基酸,与拟南芥(NP_187864.1)、番茄(NP_001266213.1)、水母雪莲花(AAB99745.1)等物种的Hsp70同源性达到80%以上,证明此基因属于Hsp70家族。根据qRT-PCR结果,高温胁迫下该基因在两个品种中的表达均上调,在耐热品种中总体表达水平均高于热敏品种,耐热品种LsHsp70-2711的表达量最大值出现在42℃、60 min,37℃、60 min时热敏品种基因表达量达最大值,且在42℃高温下热敏品种‘P-S11’中基因表达随着胁迫时间的增加受到抑制,而耐热品种‘G-S59’则能长时间保持较高的表达水平,此结果和田间两品种间耐热差异表现相符合。亚细胞定位显示,LsHsp70-2711主要在细胞质中。将构建好的载体侵入叶用莴苣,鉴定后获得阳性植株。由定量PCR结果可知,未进行胁迫处理的阳性植株与对照株相比,LsHsp70-2711表达量下降,茎长明显增长。热胁迫和干旱处理后的阳性植株LsHsp70-2711表达量显著低于对照植株,高温处理对LsHsp70-2711的影响大于干旱胁迫。【结论】LsHsp70-2711属于Hsp70基因家族,其与叶用莴苣耐热性相关。研究结果为解析叶用莴苣热激蛋白LsHsp70-2711在叶用莴苣高温抽薹方面的功能提供了理论支持。

不同红蓝LED光照强度对叶用莴苣生长和品质的影响

以叶用莴苣品种永荣为试验材料,探讨了光照强度为100μmol·m-2·s-1(RB100)、200μmol·m-2·s-1(RB200)和300μmol·m-2·s-1(RB300)的红蓝LED光源对叶用莴苣生长与品质的影响,结果显示:与其他处理相比,RB100处理叶用莴苣植株高度、叶面积、根长、比叶面积等形态指标及可溶性蛋白含量显著增加;RB300处理叶用莴苣中VC含量最高,硝酸盐和叶绿素含量最低。结果表明:300μmol·m-2·s-1光照强度有利于提高叶用莴苣的品质,而100μmol·m-2·s-1光照强度有利于叶用莴苣的生长。

叶用莴苣热激蛋白90(LsHsp90)基因的克隆及其在热激下的表达

【目的】克隆叶用莴苣热激蛋白Hsp90基因并探讨其响应热胁迫的相关机制,为揭示叶用莴苣抗热分子机制奠定理论基础。【方法】采用同源克隆和RACE技术相结合,从叶用莴苣叶片总RNA中克隆LsHsp90 cDNA序列。通过实时荧光定量PCR(qRT-PCR)分析LsHsp90在叶用莴苣耐热品种Z36和热敏品种S106受37℃和42℃热激后叶片的表达差异。【结果】该基因cDNA序列全长2 330 bp,开放阅读框2 097 bp,编码698个氨基酸,推导的蛋白质分子量约79.8 kD。蛋白质结构预测及同源比对分析表明,LsHsp90基因编码蛋白含ATPase位点和Hsp90保守结构域,并与拟南芥(AY081302.1)、紫茎泽兰(EU269070.1)等多种物种的热激蛋白90高度同源;进化树分析表明,LsHsp90与紫茎泽兰Hsp90基因(EU269070.1)聚为一类。qRT-PCR分析表明,37℃热胁迫下,该基因在耐热品种和热敏品种的叶片中均上调表达;42℃高温胁迫下,热敏品种S106中下调表达,而耐热品种Z36上调表达。【结论】成功从叶用莴苣叶片中分离克隆到LsHsp90,该基因具有已知物种Hsp90基因的特征,该基因在热胁迫下有不同的表达特征,预示该基因其可能在抗高温胁迫中起重要作用。

生菜低温胁迫转录因子LsICE1的克隆、特征分析及其转化水稻

【目的】克隆生菜(Lactuca sativa L.)低温胁迫转录因子LsICE1,对其进行序列分析和水稻遗传转化,研究超表达LsICE1基因对水稻耐低温能力的影响。【方法】设计简并引物,利用RT-PCR技术获得生菜LsICE1基因保守区域,再通过SON-PCR技术获得LsICE1基因的5′端和3′端,拼接得到全长的cDNA。对该序列进行生物信息学分析,采用半定量RT-PCR研究LsICE1的低温表达模式。最后构建植物表达载体,利用农杆菌介导法对水稻进行遗传转化。通过比较低温处理后对照和转基因株系的存活率和生理指标,鉴定超表达LsICE1基因对水稻耐低温能力的调控作用。【结果】测序结果显示,拼接后的cDNA片段长1 622 bp,包含一个1 497 bp完整的开放阅读框,编码498个氨基酸残基,命名为LsICE1,GenBank登录号为HQ848932。半定量RT-PCR研究表明,LsICE1基因是冷诱导条件下差异表达的基因。进化树分析表明,LsICE1蛋白与葡萄的ICE1蛋白亲缘关系最近,处于同一进化分枝。PCR和RT-PCR分子检测证明,LsICE1基因已经整合到水稻基因组中。与对照相比,低温处理后超表达LsICE1基因的转基因株系存活率和脯氨酸含量明显增加,相对电导率和丙二醛含量积累速率明显下降。【结论】首次从生菜中克隆了低温胁迫转录因子LsICE1,超表达LsICE1基因水稻株系提高了抗低温胁迫能力。

叶用莴苣热激蛋白LsHsp70-3701基因的克隆及高温胁迫下的表达分析

为探讨热激蛋白Hsp70基因在高温胁迫下的响应机制及其分子机理,分析Hsp70基因与叶用莴苣耐热性的关系,本研究通过同源克隆及RACE技术,克隆了叶用莴苣热激蛋白Ls Hsp70-3701基因的c DNA全长序列,利用实时荧光定量PCR(qRT-PCR)分析该基因在叶用莴苣热敏品种P-S11和耐热品种G-S59中高温胁迫下的表达差异。该基因c DNA全长为2 191 bp,开放阅读框为1 950 bp,编码649个氨基酸,具有Hsp70家族特有的标签序列,与拟南芥、番茄、小麦等物种的Hsp70同源性达到90%以上。qRT-PCR分析显示,高温胁迫下该基因在2个品种中的表达均上调,在耐热品种中总体表达水平显著高于热敏品种,且在42℃高温下热敏品种P-S11中基因表达随着胁迫时间的增加受到抑制,而耐热品种G-S59中则能长时间保持较高的表达水平。结果表明,Ls Hsp70-3701基因可能与叶用莴苣耐热性相关。本研究为有效解决叶用莴苣高温抽薹问题提供了数据支持。

甘蓝LFY基因的克隆及序列分析

以甘蓝ZQ启动抽薹的茎尖为材料,分别提取DNA和RNA,以两对引物扩增并序列拼接得到甘蓝LFYZQ基因DNA序列和cDNA完整编码区,长度分别为2560、1239bp,与花椰菜、拟南芥、芥菜和萝卜同源性分别达到91%、87%、86%、87%。该基因含有3个外显子(452、394、393bp)和2个内含子(514、807bp),共编码412个氨基酸,内含子剪接位点均符合经典GT-AG法则。将LFYZQ与网上公布的12种十字花科植物LFY氨基酸序列按分子进化分成两类:甘蓝LFYZQ与花椰菜以及Jonopsidium属植物Jonopsidium acaule这3个分为一类;其余10种植物分为第二大类。LFYZQ蛋白分子量为46kD,是一个不稳定的疏水蛋白,存在N-十四(烷)酰化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、酰胺化位点共4种活性位点。

叶用莴苣热激蛋白LsHsp70-3701和LsHsp70-1707基因与抽薹特性的表达分析

[目的]探讨叶用莴苣热激蛋白Ls Hsp70-3701和Ls Hsp70-1707基因在抽薹前后的表达水平,分析Hsp70基因与叶用莴苣抽薹的相关性。[方法]采用实时荧光定量方法检测Ls Hsp70-3701和Ls Hsp70-1707基因在不同品种中的相对表达量,分析其差异性。[结果]Ls Hsp70-3701基因在热敏品种P-S11抽薹期的表达量显著高于抽薹前;在耐热品种G-S59抽薹初期的表达量与抽薹前无显著差异,但后期表达量显著降低。Ls Hsp70-1707基因在热敏品种P-S11抽薹初期表达量与抽薹前差异不显著,但其后表达水平显著高于抽薹前;在GS59中抽薹期的最大表达量低于抽薹前的最大表达量,但两者差异不显著,而其他时间的相对表达量却显著低于抽薹前最高水平。[结论]Ls Hsp70-3701和Ls Hsp70-1707基因与叶用莴苣抽薹具有一定的相关性,叶用莴苣抽薹是差异基因表达的结果。

共转化Atpsy和folE基因提高生菜类胡萝卜素和叶酸含量

通过农杆菌介导的遗传转化法将拟南芥八氢番茄红素合成酶基因(phytoene synthase,Atpsy)和按照拟南芥偏爱密码子人工合成的编码三磷酸腺苷环化水解酶(GTP cyclohydrolase,GCHI)的folE基因共同转入罗曼生菜中,通过Realtime-PCR、HPLC、干酪乳杆菌发酵法分别测定了转基因生菜中目标基因的表达量、叶黄素和β-胡萝卜素含量以及总叶酸含量。实验结果表明:转基因生菜中Atpsy基因表达量为野生型的43.54倍,folE基因表达量为野生型的38.05倍;转基因生菜植株叶黄素和β-胡萝卜素含量与野生型相比均有提高,含量最好的转基因植株L13中叶黄素含量为7.1mg/kg(鲜重,下同),是野生型对照6.8mg/kg的1.1倍,β-胡萝卜素含量为592.6mg/kg,是野生型对照196.9 mg/kg的3倍;叶酸含量最高是2 083.4 mg/kg,为野生型对照1 129.1mg/kg的1.85倍。该研究为通过基因工程手段改良类胡萝卜素和叶酸在植物体内的代谢途径,获得同时富含类胡萝卜素和叶酸的新型生菜品种打下了基础。

叶用莴苣LsE3基因克隆与表达分析

【目的】为探明E3泛素连接酶基因对叶用莴苣高温抽薹的影响。【方法】通过RT-PCR克隆得到LsE3编码序列,生物信息学工具分析序列特征,实时荧光定量PCR方法分析基因表达水平。【结果】LsE3基因长度1 122bp,编码373个氨基酸。生物信息学分析显示,其C端有一个RING-H2finger结构域。多序列比对和系统发育分析表明,该蛋白序列具有较高的保守性,与向日葵(Helianthus annuus L.)亲缘关系较近。LsE3亚细胞定位预测位于细胞核。荧光定量PCR结果显示,高温处理显著影响植株茎中LsE3表达量。【结论】LsE3可能在叶用莴苣高温抽薹中发挥重要作用。

不同LED组合光质对莴苣灰霉病防御酶活性的影响

本试验在水培条件下,以LED红蓝光(红蓝光比例4∶1)为基础和对照,研究添加紫光、黄光、绿光组成的不同LED组合光质对叶用莴苣接种灰霉病菌后的病情指数以及接菌前后POD、PPO、PAL等相关防御酶活性的影响。结果表明,不同LED组合光质处理中,以添加紫光的LED红蓝紫处理最能降低莴苣灰霉病病情指数;防御酶活性上,红蓝光基础上添加紫光和黄光处理的POD、PAL、PPO、GLU活性较高,添加绿光、黄光处理的CHT活性较高。整体上看,在LED红蓝光基础上添加一定比例的紫光或黄光,不仅可以有效降低莴苣灰霉病的病情指数,还可以提高莴苣体内的防御酶活性。该结果可为莴苣生产中通过光环境控制来抑制灰霉病发生提供理论参考。

Obtaining and Cytological Identification of a Set of Primary Trisomics in Cabbage

Selection of primary trisomics of the cabbage （Brassica oleracea var.capitata L） forms an important basis for gene chromosome mapping and for other genetic studies. The cabbage self-fertilization line - 9601 was used as material, using the root-tip cell chromosome number and pollen mother cell chromosome number identification and karyotype analysis to select the primary trisomics from the progenies of 3x x 2x in the cabbage. Many aneuploid plants with one or two extra chromosomes were obtained and a set of primary trisomics （Tri-1, Tri-2, Tri-3, Tri-4, Tri-5, Tri-6, Tri-7, Tri-8, and Tri-9, in which the Tri-1 and Tri-4 were from 2n＋2 plants and others from 2n＋ 1 plants） was acquired from these plants. Each trisomic exhibited some unique features, such as plant height, plant type, leaf type, size of flower bud, and inflorescence. The triploid crossing by the diploid is a convenient and effective way to select trisomics in the cabbage.