Quercetin-derivatives painting the yellow petals of American lotus(Nelumbo lutea)and enzymatic basis for their accumulation

American lotus(Nelumbo lutea)is one of the two species in Nelumbo and has only yellow flower.Identification of total flavonoids showed wild American lotus contained almost only flavonols with quercetin 3-O-glucuronide to be the dominant pigment.The variation tendency of the total flavonol content was coincident with yellow color variation of petals during flower development.To understand the mechanism of accumulation and constituent of pigments in petals,three pivotal genes,NlFLS1,NlFLS2 and NlFLS3,which were predicted to encode flavonol synthases were isolated and characterized by analyses of basic bioinformatics,temporal and spatial expression patterns and enzymatic activity.Their temporal expression levels showed the same variation tendency,which was also consistent with the development-dependent variation of total flavonol content.Spatial expression patterns indicated the three genes should function in petals.All the three proteins were demonstrated to be bifunctional dioxygenase,possessing both flavonol synthase activity and flavanone 3-hydroxylase activity.Besides,other flavonol biosynthesis related genes were also investigated on their expression levels to give more clues on the mechanism.Substrate preferences of the three FLSs,substrate competitions between the FLSs and other flavonol biosynthesis related enzymes,and the greatly differential expression levels between F3’H(flavonoid 3-hydroxylase)and F3’5’H(flavonoid 3,5-hydroxylase)contributed to the flavonol constituent in the petals of America lotus,namely abundant quercetin-derivatives while very few kaempferol-derivatives and myricetin-derivatives.

Genetic diversity and inferred ancestry of Asian lotus(Nelumbo nucifera) germplasms in Thailand and Vietnam

Tropical lotus(Nelumbo) is an important and unique ecological type of lotus germplasm.Understanding the genetic relationship and diversity of the tropical lotus is necessary for its sustainable conservation and utilization.Using 42 EST-SSR(expressed sequence tag-simple sequence repeats) and 30 SRAP(sequence-related amplified polymorphism) markers,we assessed the genetic diversity and inferred the ancestry of representative tropical lotus from Thailand and Vietnam.In total,164 and 41 polymorphic bands were detected in 69 accessions by 36 EST-SSR and seven SRAP makers,respectively.Higher genetic diversity was revealed in Thai lotus than in Vietnamese lotus.A Neighbor-Joining tree of five main clusters was constructed using combined EST-SSR and SRAP markers.Cluster Ⅰ included 17 accessions of Thai lotus;cluster Ⅱ contained three Thai accessions and 11 accessions from southern Vietnam;and cluster Ⅲ was constituted by 13 accessions of seed lotus.Consistent with the results from the NeighborJoining tree,the genetic structure analysis showed that the genetic background of most Thai and Vietnamese lotus was pure,as artificial breeding has been rare in both countries.Furthermore,these analyses indicate that Thai and Vietnamese lotus germplasms belong to two different gene pools or populations.Most lotus accessions are genetically related to geographical distribution patterns in Thailand or Vietnam.Our findings showed that the origin or genetic relationships of some unidentified lotus sources can be evaluated by comparing morphological characteristics and the data of molecular markers.In addition,these findings provide reliable information for the targeted conservation of tropical lotus and parent selection in breeding novel cultivars of lotus.

A Review on the Taxonomic,Evolutionary and Phytogeographic Studies of the Lotus Plant(Nelumbonaceae:Nelumbo)

Nelumbo Adans. （Nelumbonaceae） is an important member of the early-diverging eudicots. It contains two extant species： N. nucifera Gaertn. （the Sacred lotus）, distributed in Asia and Australia and N. lutea Willd. （the American lotus）, occurring in North America. This paper reviews the taxonomic, evolutionary and phytogeographic studies of the genus Nelumbo, and also raises scientific questions about it in further paleobotanic research. There are about 30 fossil species of Nelumbo established since the Early Cretaceous. Based on fossil studies, the ancestors of the extant N. nucifera and N. lutea are respectively considered to be N. protospeciosa from the Eocene to Miocene of Eurasia, and N. protolutea from the Eocene of North American. However, molecular systematic studies indicate that N. nucifera and N. lutea are probably split from a common ancestor during the Late Miocene to Early Pliocene, or even the Pleistocene, rather than separate relicts from extinct species on different continents. The characters of lotus stomatal development, seedling morphology as well as its flowering, pollination and fertilization in air reveal that it evolves from the land plants. Fossil data of Nelumbo indicates that the genus first occurs in mid-latitude area of Laurasia in the Early Cretaceous, then becomes widespread in North America and Eurasia and expands into Africa and South America during the Late Cretaceous; the genus probably colonizes the Indian Subcontinent from Asia during the Early Eocene after the collision of India and the Asian plates; the genus becomes extinct in Europe, but survives in Asia and North America during the Quaternary Ice Age, and later forms the present East Asia and North Australia-North America disjunctive distribution.

中国莲(Nelumbo nucifera)幼苗抗氧化系统对砷胁迫的响应

为了研究中国莲(Nelumbo nucifera)抗氧化系统对砷胁迫的响应,研究比较了两种不同价态无机砷As(Ⅲ)和砷As(Ⅴ)对中国莲幼苗可溶性蛋白含量、丙二醛(MDA)含量以及抗氧化系统的影响。结果表明,中国莲幼苗中MDA和可溶性蛋白质含量随砷浓度的增加呈现先升高后降低的趋势。中国莲幼苗的MDA和蛋白质含量受As(Ⅴ)的影响不如As(Ⅲ)敏感。抗氧化系统酶中,超氧化物歧化酶(SOD)对砷处理最敏感,当As(Ⅲ)浓度在2.5μmol/L和As(Ⅴ)浓度在100μmol/L时,SOD酶活性显著高于对照组。过氧化物酶(POD)在As(Ⅲ)处理浓度为10μmol/L时就出现显著上升,相对而言,过氧化氢酶(CAT)对As(Ⅴ)比较敏感。实验结果表明,随着浓度的增加,砷对幼苗产生的氧化胁迫导致SOD、CAT和POD三种酶活性有所增加,以配合清除细胞内的活性氧自由基(ROS),维持细胞代谢的稳定。本研究为进一步研究砷胁迫下莲的生理和生长变化、以及莲的培育和移植提供了部分基础数据。

莲属(Nelumbo Adans.)的系统学研究进展和莲科的确立

本文介绍了莲属的系统学研究的历史和最新研究成果,综述了莲属从睡莲科中独立出来,单独成科的形态学、解剖学、细胞学、孢粉学、胚胎学、植物化学、分子生物学及花器官发生等多方面的证据,并对莲科的系统学地位进行了总结。

A new benzylisoquinoline alkaloid from stems of Nelumbo nucifera

A new benzylisoquinoline alkaloid, named nelumstemine （1）, 1-（4＇-hydroxybenzoyl）-6,7-dimethoxy-3,4-dihydroisoquinoline, was isolated from the stems of Nelumbo nucifera Geartn. Its structure was established on the basis of spectral analysis.

Chromosomal Localization of the 5S and 45S rDNA Sites and 5S rDNA Sequence Analysis in Nelumbo Species

The physical location of 45S and 5S rDNA sites by double-target fluorescence in situ hybridization （FISH） and 5S rDNA non-transcribed spacer （NTS） sequences was studied in five accessions of Nelumbo nucifera Gaertn.and one accession of N.nucifera subsp.lutea （Willd.）.The chromosome number of all tested materials was 2n=16.The loci of 5S rDNA were close to the centromere regions of chromosome 3,and the loci of 45S rDNA were found on chromosomes 7 and 8 in all the six tested accessions,respectively.Similarly,45S rDNAs were also located on chromosome 4 in four tested accessions.The 5S rRNA gene sequences were conserved and the NTS sequences were variable among the samples.N.nucifera subsp.lutea was the longest branch in the phylogenetic tree and clustered with N.nucifera cv.Liangzihu.N.nucifera cv.Heilongjiang and N.nucifera cv.Weishanhu showed a close relationship with each other.N.nucifera cv.Dahe Lian was closer to N.nucifera cv.Nihelu Lian than to other tested accessions.Analysis of the molecular karyotype and the 5S rRNA gene spacer sequence suggested the genetic diversity was limited within Nelumbo species and it seems suitable that American lotus was considered as the subspecies of N.nucifera.

Analysis of Accumulating Ability of Heavy Metals in Lotus (Nelumbo nucifera) Improved by Ion Implantation

Heavy metals have seriously contaminated soil and water, and done harm to public health. Academician WANG Naiyan proposed that ion-implantation technique should be exploited for environmental bioremediation by mutating and breeding plants or microbes. By implanting N^＋ into Taikonglian No.l, we have selected and bred two lotus cultivars, Jingguang No.1 and Jingguang No.2. The present study aims at analyzing the feasibility that irradiation can be used for remediation of soil and water from heavy metals. Compared with parent Taikonglian No.l, the uptaking and accumulating ability of heavy metals in two mutated cultivars was obviously improved. So ion implantation technique can indeed be used in bioremediation of heavy metals in soil and water, but it is hard to select and breed a cultivar which can remedy the soil and water from all the heavy metals.

Genetic diversity of the wild Asian lotus (Nelumbo nucifera) from Northern China

Asian lotus(Nelumbo nucifera Gaertn.)is an aquatic plant with ornamental,cultural,economic,and ecological values.China has abundant germplasm resources of Asian lotus.However,in many areas,the wild Asian lotuses have been destroyed and the germplasms is now facing extinction.In addition,the knowledge of the genetic diversity of the wild Asian lotus in China is poor.To identify and protect the germplasms of Chinese wild Asian lotus,eleven genomic-SSR primers,three EST-SSR primers,and three chloroplast DNA primers were used to investigate the genetic diversity among 69 samples of wild Asian lotus from 25 locations in northern China.The genetic diversity of 27 samples of wild Asian lotus from southern China and other countries,the ancient Asian lotus,Asian lotus cultivars from China,and Asian-American hybrids was also compared.The genetic diversity of the wild Asian lotus from northern China was characterized as medium,and themean values of observed heterozygosity(Ho)and expected heterozygosity(He)were 0.087 and 0.552,respectively.Based on a UPGMA dendrogram and STRUCTURE analysis,the wild Asian lotus samples in northern China were divided into three groups.The wild Asian lotus samples from northern China contained 16 haplotypes.The Nei’s genetic distance between the wild Asian lotus samples from the Songhua River basin and the Liao River basin in northeastern China was relatively small,and these germplasms might be relatively primitive compared to those from other regions.This study provides essential information regarding the genetic diversity of the wild Asian lotus resources in northern China,and provides a basis for further analysis of population-level genetic evolution through high-throughput sequencing.

Gibberellins,brassinolide,and ethylene signaling were involved in flower differentiation and development in Nelumbo nucifera

Hormones play important roles in vegetative and reproductive processes;however,the regulatory roles of hormones in Nelumbo nucifera(Lotus)growth and development are unclear.In this study,nine types of endogenous hormones,including gibberellins(GA_1,GA_3,and GA_4),indole-3-acetic acid(IAA),brassinolide(BR),ethylene(ETH),jasmonic acid(JA),abscisic acid(ABA),and zeatin(ZT)were detected in dormant shoot tips,vegetative shoot tips,developing leaf buds,and developing flower buds of lotus.The results indicated that GA,ETH,and BR signaling can promote vegetative and reproductive development of lotus.GA signaling regulates plant height and stimulates flower bud differentiation.GA levels were the highest in the flower buds;exogenous GA3+4 increased plant height by approximately 90%,increased flower quantity by nearly 40%,and advanced flowering by 4 d.Suppressing GA biosynthesis using paclobutrazol decreased plant height and flower quantity by 38%and 87.1%,respectively,and delayed flowering by 15.6 d.ETH signaling has positive regulatory effects on vegetative growth and flower development.The ETH concentration in the developing leaf buds was at least 50%higher than that in other samples.Ethephon spraying led to remarkable increases in plant height and leaf thickness and extended the flowering duration.BR signaling acts as a growth promoter during vegetative and reproductive development in lotus.The highest BR levels were detected in the vegetative shoot tips.External application of28-epihomobrassinolide resulted in growth-promoting phenotypes including longer scapes,thicker leaves,and prolonged flowering.

梁山古莲(Nelumbo nucifera Gaertn.)的种质资源价值研究

对2013年梁山出土的古莲(命名为梁山古莲)进行了种质资源价值研究。断代分析结果显示,梁山古莲籽距今时间最长约为731 a,入土年代处于我国元朝初期,古莲籽发芽率在90%以上,不同方位取得的莲籽大小存在明显差异;表型分析表明,梁山古莲属于中国莲系大株型少瓣红莲类群,为黄河流域野生莲,古莲群体存在较高的遗传多样性;聚类分析显示,梁山古莲与山东地方品种马踏湖白莲亲缘关系最近。

Genetic Diversity of Lotus(Nelumbo nucifera Gaertn.) Cultivars Revealed by ISSR Analysis

[ Objectives ] This study was conducted to investigate the genetic diversity of 20 lotus （Nelumbo nucifera Gaertn） samples. [ Methods ] On this optimal ISSR amplification system, 16 primers were screened with good polymorphism, and the DNA was used to amplify the 20 plant samples. [ R^ults] The 16 primers produced 225 loci, of which 170 were polymorphic, and the polymorphic loci percentage was up to 75.56%. The genetic similarity coefficients between the 20 vari- eties ranged from 0. 577 8 to 0.951 1, which were calculated by POPGENE32. The 20 varieties by UPMGA analysis could be clustered into 2 groups, the first of which included Baiyangdian red lotus and Donggua lotus, and other varieties was included in the second group. [ Conchmions] ISSR molecular markers could be effectively used in genetic diversity and fingerprint analysis for different lotus varieties.

Genetic Variation of the Novel Badnaviruses Infecting Nelumbo Nucifera Based on the RT/RNase H Coding Region Sequences

Lotus(Nelumbo nucifera Gaertn.)is an economically important perennial aquatic herb plant in China.Presently,viral diseases caused by Cucumber mosaic virus(CMV)and Dasheen mosaic virus(DsMV)are detected in lotus.In this study,the putatively novel badnaviruses from lotus plants(LBVs)were identified using next-generation sequencing of siRNAs and conventional Sanger sequencing based on the RT/RNase H coding region sequences.Lotus plants infected by LBVs harbored virus quasispecies.A unique recombination event of LBVs was found in a single lotus plant.Sap inoculation showed that LBVs could actively replicate in the lotus plants,but without a wide host range.The field survey of 43 lotus plants in Jiangsu Province showed a prevalence of 62.8%for LBVs,confirming that it is widely distributed in Jiangsu Province of China.

Evaluation of the Anti-Hyperlipidemic Activity of <i>Nelumbo nucifera </i>Fruit in Rabbits Fed with High Cholesterol Diet

Recently use of herbal therapies and diet rich in flavonoids and vitamins has augmented significantly to manage minor to modest illnesses. Several anti-hyperlipidemic agents are currently available;however most of them have various unwanted effects. That’s why more people are switching towards safer alternatives, specially derived from plants with fewer side effects. Hence current study was focused to determine the anti-hyperlipidemic potential of Nelumbo nucifera fruit in rabbits fed with high cholesterol diet. This study would surely help in the exploration of a novel agent from the plant source and therefore may recommend the application of traditional medicines in humans. 28 white healthy rabbits of either sex weighing 1000 - 1500 gm were divided into four groups. Control group was given gum tragacanth and 3 groups served as test groups and were given N. nucifera fruit extract 50, 100 and 200 mg/kg for 45 days. Initially animals of all four groups received high cholesterol diet (HCD) 0.125 gm/kg cholesterol in 0.5% corn oil for 30 days. All drugs were given orally. Blood samples were taken thrice from the ear vein of animals, 1st after 24 hours of thirty days of HCD then again after 24 hours of thirty and forty five days dosing of N. nucifera fruit extract. The results showed remarkable cholesterol lowering effects of N. nucifera fruit extract at doses 50, 100 and 200 mg/kg but were particularly dominant at 200 mg/kg at which fruit extract highly significantly reduced TC, TG and LDL-C after 30 and 45 days and increased HDL-C significantly after 30 days and highly significantly after 45 days. Flavonoids, saponins, tannins and procyanidin are significant constituents of N. nucifera fruit which may have contributed in lipid lowering effects of N. nucifera fruit which can contribute in lowering the risk of CVDs and stroke but more preclinical and clinical studies are required to confirm these findings.

国内外8个不同种源地莲(Nelumbo)的成熟胚离体培养比较

建立高效获取无菌莲胚外植体的消毒体系,了解不同种源地莲的离体培养生长差异,为今后构建莲的优良离体培养及遗传转化体系提供可靠的材料和评估方法。使用不同浓度植物保护混合剂(plant preservative mixture,PPM)预培养法确定适宜的莲子消毒体系和无菌莲胚获取方式;在此基础上获得国内外9个种源地莲的成熟胚离体培养幼苗,通过观测其在不同培养时期的形态指标,利用隶属函数法综合评价其离体条件下的生长差异。研究发现,莲子比较理想的消毒体系为75%酒精消毒30 s+2%PPM消毒2 h+0.05%PPM浸泡催芽3~5天。其中,8个参试种源地莲的离体生长差异主要体现在培养后期,表现在展开叶数、茎节长度和粗度、根数及侧芽数等指标上。在相同培养条件下,越南莲和泰国莲的生长势最好、繁殖率较高。使用PPM浸泡成熟莲子进行预培养以获取无菌莲胚的方式,克服了以往莲子消毒不理想的技术难题。热带、亚热带分布的越南莲和泰国莲的离体无性系生长势强,是构建莲再生体系和遗传转化体系的良好原材料。

采用AFLP技术分析17个荷花(Nelumbo nucifera)品种的系统关系

【目的】采用AFLP技术,对17个荷花品种进行亲缘关系分析。【方法】从64个AFLP引物组合中筛选出10对多态性较高的引物,以17个荷花DNA为模板,进行PCR扩增,并对产物进行聚丙烯凝胶电泳分析。【结果】共获得条带523条,其中多态性条带370条,多态性比例达到70.75%;根据扩增条带信息进行聚类,可将17个品种分为2组;其中有5对引物,每对引物单独使用均可将17个品种完全区分开。【结论】AFLP用于鉴别荷花品种,具有较高的可行性;本研究为荷花分类和杂交育种的亲本选择提供参考信息。

莲属植物(Nelumbo spp.)在美国东南部的利用潜力研究

莲(Nelumbo spp)的利用为黑土带经济开发提供了一个独特的低成本和低风险机遇。对鲢鱼塘稍加改造,莲即可与鲢鱼实行搭配种养。对160多个莲品种的研究,使得花莲的大量生产销售成为可能。1999年以来,还一直致力于花莲栽培的"最优管理规范"开发。目前,正对"莲-鱼"搭配种养、莲对营养物和有机物的植物修复潜能进行研究评价。对莲的有关研究结果进行了总结。

荷花(Nelumbo nucifera)全基因组PRR基因家族鉴定及其在多种非生物胁迫下的表达模式

为了探究荷花(Nelumbo nucifera)伪应答调控蛋白(pseudo-response regulators, PRR)NnPRR家族成员的特征及其在非生物胁迫响应中的作用,通过生物信息学分析,将7个NnPRR基因从荷花全基因组中挖掘出来,对其基因结构、蛋白保守基序、顺式作用元件及共线性关系等进行了鉴定分析,并利用实时定量PCR技术探究各基因分别在外源施加300 mmol/L NaCl、 5 mmol/L水杨酸(salicylic acid, SA)及4℃低温处理下的表达模式。研究结果表明,该家族基因在荷花中可分为3个分支,集中分布在chr2、chr3、chr5和chr6染色体上,该家族蛋白均定位于细胞核。该家族基因在启动子区域发现大量光响应元件,且一些基因还具有低温、茉莉酸甲酯及脱落酸等响应元件。从基因的表达模式上看,NnPRR基因家族7个基因均正向响应外源NaCl处理,而NnPRR2、NnPRR4、NnPRR3等基因则特异性响应外源SA或低温胁迫,推测该家族基因特异性参与荷花非生物逆境胁迫。本研究结果为深入研究荷花PRR基因家族响应逆境胁迫提供了理论基础,也为抗逆新品种的选育提供了新的基因资源。

美洲莲地下茎顶芽离体培养

以美洲莲地下茎顶芽为外植体,主要对消毒处理方法、顶芽初代培养激素种类及其浓度进行摸索,并比较取材时间和培养温度对顶芽离体培养成苗的影响。研究表明:对顶芽采取消毒后去掉一层鳞片再消毒,或在此基础上再去掉一层鳞片,将去掉一层或两层鳞片后的剩余部分接种至含有0.1%植物组培抗菌保护剂(Plant preservative mixture,PPM)或250 mg∕L头孢霉素的培养基中培养,污染率能降低到10.71%—15.99%。外植体在含3 mg∕L激动素(Kinetin,KT)+0.2 mg∕L萘乙酸(1-Naphthaleneacetic acid,NAA)的MS(Murashige and Skoog)培养基上培养1个月后转移至含1 mg∕L KT+0.1 mg∕L NAA的培养基上,或始终在含2 mg∕L KT+0.2 mg∕L NAA培养基上培养,能获得较好的成苗和增殖效果。10—12月份冬季休眠期的芽比3—5月份春季萌芽期至营养生长前期的芽更易培养成苗。30℃较25℃培养更利于顶芽的成苗。该研究建立的美洲莲地下茎顶芽离体培养体系可为该物种的推广应用以及试验研究提供一致性的材料和技术支持。

山东莲藕(Nelumbo nucifera Gaertn.)种质资源的RAPD鉴定和遗传多样性分析

山东莲藕(Nelumbo nucifera Gaertn.)栽培历史悠久,在中国北方具有重要影响。但是,山东莲藕种质资源研究一直没有开展。为收集和保存莲藕种质资源,本研究从山东省8个种植面积较大的地区采集了76份莲藕材料,并利用RAPD分子标记对这些材料进行了遗传相似性分析,共鉴定出38份种质资源。遗传多样性指数分析显示,38份种质资源的Nei's多样性指数(H)平均值为0.26,Shannon信息指数(I)平均值为0.41。研究数据表明,山东莲藕种质资源相对丰富,但要建立高质量的种质资源圃,仍需要加强种质资源的引进。