Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes (FBEs) in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-（2,1）- and β-（2,6）-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes （sucrose： sucrose 1-fructosyltransferase, 1-SST, EC： 2.4.1.99; sucrose： fructan 6-fructosyltransferase, 6- SFT, EC： 2.4.1.10; and fructan： fructan 1-fructosyltransferase, 1-FFT, EC： 2.4.1.100） were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat （Triticum turgidum, genotype： AABB）, common wheat （Triticum aestivum L., genotype： AABBDD） and three wild relatives of common wheat, Triticum urartu Thum. （the origin of the AA genome）, Aegilops speltoides （Tausch） Gren. （the putative source of the SS genome） and Aegilops tauschii Coss. （the source of the DD genome）. Sequence analysis revealed that all the FBEs （fructan biosynthetic enzymes） had three highly conserved functional motifs except 1-SST （EU981912） from tetraploid wheat species only with conserved DPNG. Low pI （isoelectric point） and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1-SST and 1-FFT were more conservative and had closer relationship each other, while 6-SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.

Tissue Nitrogen and Fructan Translocation in Bread Wheat

Translocation of previously accumulated nitrogen and carbohydrates from vegetative tissue of the wheat plant is a major assimilate source for grain filling. This study was conducted to examine genotype differences in nitrogen and fructan translocation and their relationships to grain yield and protein content. Effects indicated that significant genotype differences existed for nitrogen accumulation at anthesis and fructan at milk stage and their translocation. Two high protein genotypes, Cunningham and PST90-19, accumulated more nitrogen before anthesis and had greater nitrogen translocation, but lower post-anthesis nitrogen uptake, than two low protein genotypes, SUN109A and TM56. Among plant parts, leaves were the major storage for tissue nitrogen and provided the overwhelming proportion of the total nitrogen translocation, whereas for fructan accumulation and translocation it was the stems. The two high protein genotypes had a higher percentage of their grain nitrogen derived from nitrogen translocation, while for the two low protein ones, it was from post-anthesis nitrogen uptake and assimilation. Increasing nitrogen application increased nitrogen accumulation and translocation, but decreased fructan accumulation and translocation. High grain protein content was associated with high nitrogen translocation from leaves, stems and the total plant, while high grain yield was related to high fructan translocation from stems and the total plant. Fructan translocation was negatively correlated to grain protein content. Nitrogen and fructan translocation were not correlated with each other.

Study on prebiotic effectiveness of neutral garlic fructan in vitro

Garlic is traditionally believed to have many health benefits including prevention of gastrointestinal diseases.One of the major components,garlic fructan(GF),was evaluated for its prebiotic effectiveness on human intestinal microflora.Garlic fructans A(DP 16)and B(DP 21)were prepared by ethanol fractionation precipitation.Then,they were added to an in vitro fermentation system as the sole carbon source inoculated with human fecal suspension.The total anaerobic bacteria,Bacteroides and Bifidobacteria,were enumerated by plate counting on selective media.Terminal restriction fragment length polymerization(tRFLP)was used to analyze DNA extracted from the in vitro cultures.The results indicated that the log CFUs of both Bacteroides(GF A 6.96,GF B 7.15)and Bifidobacteria(GF A 7.74,GF B 7.74)grown in the GF cultures at 24 h were significantly higher than those at 0 h(Bacteroides 4.93,Bifidobacteria 4.78)(P<0.05).Terminal restriction fragments(TRFs)256–258 bp(ascribed to Bifidobacterium)in the profiles were also observed higher in the TRFLP profiles from the garlic fructans’cultures.In this study,GFs were found to selectively stimulate the growth of beneficial Bifidobacteria from human fecal microflora.The prebiotic effectiveness of GFs supports the use of garlic as a way to prevent some gastrointestinal diseases.©2013 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.All rights reserved.

Relationship of Grain Fructan Content to Degree of Polymerisation in Different Barleys

Fructans are important in the survival of plants and also valuable for humans as potentially health promoting food ingredients. In this study fructan content and composition were determined in grains of 20 barley breeding lines and cultivars with a wide variation in chemical composition, morphology and country of origin, grown at one site in Chile. There was significant genotypic variation in grain fructan content ranging from 0.9% to 4.2% of grain dry weight. Fructan degree of polymerisation (DP) was analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Changes in the distribution of different chain lengths and the pattern of structures of fructan were detected with increasing amount of fructan in the different barleys. A positive correlation was found between fructan content and the relative amount of long chain fructan (DP > 9) (r = 0.54, p = 0.021). Our results provide a basis for selecting promising barley lines and cultivars for further research on fructan in barley breeding with the aim to produce healthy food products.

Acute Toxicity and Genotoxic Evaluation of Metlin^®and Metlos^®(Organic Agave Fructans)

The aim of the study was to contribute to the information on agave soluble fibers since research has been focused on chicory fiber but not in agave products;thus we assess the acute toxicity and genotoxicity of two organic and high purity dietary soluble fibers from agave, Metlin? and Metlos?. We performed an acute toxicity assay in Hsd:ICR mice and Hsd:Wistar rats and an in vivo genotoxic test. Results showed that there are no deaths at any doses or genotoxicity, so it can be concluded that these products are non-toxic, at the administrated doses and none showed a cytotoxic, clastogenic or aneuploidic effect.

An inulin-type fructan CP-A from Codonopsis pilosula attenuates experimental colitis in mice by promoting autophagy-mediated inactivation of NLRP3 inflammasome

Inulin-type fructan CP-A,a predominant polysaccharide in Codonopsis pilosula,demonstrates regulatory effects on immune activity and anti-inflammation.The efficacy of CP-A in treating ulcerative colitis(UC)is,however,not well-established.This study employed an in vitro lipopolysaccharide(LPS)-induced colonic epithelial cell model(NCM460)and an in vivo dextran sulfate sodium(DSS)-induced colitis mouse model to explore CP-A’s protective effects against experimental colitis and its underlying mechanisms.We monitored the clinical symptoms in mice using various parameters:body weight,disease activity index(DAI),colon length,spleen weight,and histopathological scores.Additionally,molecular markers were assessed through enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qRT-PCR),immunofluorescence(IF),immunohistochemistry(IHC),and Western blotting assays.Results showed that CP-A significantly reduced reactive oxygen species(ROS),tumor necrosis factor-alpha(TNF-α),and interleukins(IL-6,IL-1β,IL-18)in LPS-induced cells while increasing IL-4 and IL-10 levels and enhancing the expression of Claudin-1,ZO-1,and occludin proteins in NCM460 cells.Correspondingly,in vivo findings revealed that CPA administration markedly improved DAI,reduced colon shortening,and decreased the production of myeloperoxidase(MPO),malondialdehyde(MDA),ROS,IL-1β,IL-18,and NOD-like receptor protein 3(NLRP3)inflammasome-associated genes/proteins in UC mice.CP-A treatment also elevated glutathione(GSH)and superoxide dismutase(SOD)levels,stimulated autophagy(LC3B,P62,Beclin-1,and ATG5),and reinforced Claudin-1 and ZO-1 expression,thereby aiding in intestinal epithelial barrier repair in colitis mice.Notably,the inhibition of autophagy via chloroquine(CQ)diminished CP-A’s protective impact against colitis in vivo.These findings elucidate that CP-A’s therapeutic effect on experimental colitis possibly involves mitigating intestinal inflammation through autophagymediated NLRP3 inflammasome inactivation.Consequently,inulin-type fructan CP-A emerges as a promising drug candidate for UC treatment.

Structural Features of Fructans from the Root of Cyathula officinalis Kuan

Three fructans (CoPS1, CoPS2 and CoPS3) vrere isolated from the root of Cyathula officinalis Kuan, a traditional Chinese medicine. The structures of the fructans were determined by methylation, reductive-cleavage method combined with GC-MS analysis, and 13C NMR spectroscopy. These results show that the fructans (CoPS1, CoPS2 and CoPS3) are graminan type fructans, and comprised of (2→1)- and (2→6)-linked β-D-fructofuranosyl backbone residues containing high branches.

Structural study on a bioactive fructan from the root of Achyranthes bidentata Blume

The structure of a saccharide component (Abs), with pronounced activity of improving immunity system, isolated from the root of Achyranthes bidentata Blume, a traditional Chinese herbal medicine, was studied. Based on C-13 NMR, HPLC, and methylation analyses, Abs was shown to be a mixture of short-chain fructans with an average dp of 8, containing more (2-->B) than (2-->1) linked beta-D-fructofuranosyl residues, with branching at O-6 or O-1 of 18% of the D-fructofuranosyl residues.

适度土壤干旱对贪青小麦茎贮藏碳水化合物向籽粒运转的调节

以春性小麦品种扬麦15 8和扬麦11为材料,设置出穗后高氮(HN)和正常氮(NN)两个施肥水平,以正常浇水为对照(WW) ,研究适度土壤干旱(WS)对小麦茎贮藏性碳水化合物运转及有关酶活性的影响。结果表明,土壤干旱改变了小麦茎中碳水化合物的组成和流向,通过诱导小麦茎节间果聚糖外水解酶(FEH)活性的上升,促进了贮藏果聚糖的降解。适度土壤干旱抑制蔗糖代谢过程中由蔗糖合成酶(SuSy)催化的可逆途径,但加强蔗糖磷酸合成酶(SPS)和蔗糖转化酶(INV)催化的单向碳流途径,SPS在茎内蔗糖的合成中起主导作用,促进了开花期喂入的贮藏14 C向蔗糖的分配,在茎贮藏同化物快速运转期(10～30DAA) ,土壤干旱处理使小麦茎中蔗糖含量较正常浇水处理分别提高了15 .9%～19. 0 %(NN)和34. 7%～4 0 . 5 % (HN) ,贮藏14 C向籽粒分配的比例显著提高,茎鞘贮藏性糖对籽粒产量贡献分别是对照(正常浇水处理)的2 . 0～2 . 1倍(NN)和4 . 8～5 . 3倍(HN)。与正常浇水处理相比,土壤干旱使正常施氮处理的籽粒产量降低,而使高氮处理的产量显著增加。

羊草根茎的贮藏碳水化合物及对氮素添加的响应

为了研究氮素对羊草 (Leymuschinensis)根茎碳水化合物贮藏的影响 ,在中国科学院内蒙古草原生态系统定位研究站的羊草样地 ,设计了不同水平和不同时期的氮素添加试验。采用高效液相色谱 (HighPerformanceLiquidChromatography ,HPLC)对羊草根茎中的贮藏性碳水化合物进行了测定。结果表明 ,羊草根茎中的贮藏碳水化合物组分包括果聚糖、甘露糖醇、蔗糖、葡萄糖和果糖。其中果聚糖是最主要贮藏碳水化合物 ,约占 6 0 % ;其次是甘露糖醇 ,约占 2 0 %。氮素添加量对羊草根茎中的贮藏碳水化合物有显著影响。在 0～ 17.5gN·m-2 范围内 ,随着氮素添加量的增加 ,碳水化合物总量、果聚糖、甘露糖醇的含量均逐渐升高。氮素添加时期对羊草根茎中的贮藏碳水化合物的含量亦有显著影响。在 7月初添加氮素比

植物非结构性贮藏碳水化合物的生理生态学研究进展

非结构性碳水化合物是参与植物生命过程的重要物质。蔗糖不仅是植物体内碳水化合物运输的主要形式 ,而且可以在基因表达水平上对细胞内的代谢进行调节。果聚糖是植物营养组织碳水化合物的主要暂贮形式 ;淀粉是植物主要的长期贮存物质之一。植物体内非结构性碳水化合物的代谢在很大程度上影响着植株的生长发育和对环境因子的响应。综述了植物非结构性贮藏碳水化合物的生理生态学研究进展 ,着重介绍了蔗糖、果聚糖和淀粉代谢的生理过程及对环境因子 (温度和水分 )和人为因素的响应机制。

几个小麦基因型苗期抗旱性鉴定及相关生理指标分析

为了明确小麦抗旱性与果聚糖含量和叶绿素荧光特性间的关系，选用扬麦6号、扬麦12、新春9号、Bobwhite、宁春27、CB9945、03S58、8139等8个小麦基因型，从分蘖期开始进行干旱胁迫处理，每隔一定时期取叶片测定果聚糖含量，36d后复水，统计存活率，抽穗期测定旗叶叶绿素荧光参数。结果表明，扬麦6号干旱胁迫处理后植株存活率（60．71％）显著高于其他几个基因型（1．19％～28．57％），表现出较强的抗旱性；干旱胁迫期间，扬麦6号叶片中果聚糖含量明显增加，而其他基因型叶片中果聚糖含量几乎没有变化；扬麦6号PSⅡ原初光能转换效率（Fv／Fm）最高（0．7977），显著高于03S58（0．6932）、Bobwhite（0．6879）和宁春27号（0．6285）；PSⅡ潜在活性（Fu／Fo）也以扬麦6号最高（3．9484），显著高于Bobwhite（2．7303）、新春9号（2．7187）、03S58（2．4034）和宁春27号（2．1619）。这说明小麦苗期抗旱性与果聚糖含量和叶绿素荧光特性间有一定的关系，抗旱性强的基因型干旱胁迫期间果聚糖含量、叶绿素荧光参数Fv／Fm均较高。

盐胁迫对菊芋干物质和糖分积累分配的影响

中国存在大量盐碱地，通过筛选耐盐植物可以改造部分盐碱地，但盐害通常极大的影响了植物的正常生长，菊芋因为耐旱、耐盐，病虫害少，可简化栽培，已经日益受到广泛重视。在本研究中以菊芋品种南芋一号（ＮＹ-１）和青芋二号（ＱＹ-２）两种品种为试材，在温室进行全生育期土培盆栽试验，研究了盐胁迫对菊芋干物质和糖分积累分配的影响。结果表明，盐胁迫改变了两个菊芋品种的干物质分配格局，地上部分配比例均增大而块茎分配比例均减小，对ＱＹ-２的限制作用比ＮＹ-１更大，盐胁迫下ＮＹ-１和ＱＹ-２块茎干重降幅分别为５７．７８％和８５．６１％。在持续的盐处理下，两个品种的块茎和茎中的可溶性糖含量均降低，但ＮＹ-１的含量比ＱＹ-２高；两个品种的茎中还原糖含量下降幅度较大，降幅分别为ＮＹ-１（７７．３４％）和ＱＹ-２（６０．７９％），但茎中的非还原性糖含量却有所增加。盐处理使块茎发育前期（１００～１４０ｄ，ｄａｙｓａｆｔｅｒｐｌａｎｔｉｎｇ）的果聚糖含量上升，ＮＹ-１在１４０ｄ的果聚糖含量显著高于对照，ＱＹ-２在１００～１４０ｄ的果聚糖含量显著高于对照，但降低了块茎发育中后期（１４０～２２０ｄ）的果聚糖含量。盐胁迫对菊芋块茎中果聚糖积累的限制作用极其显著，尤其对ＱＹ-２的限制作用尤为突出，ＱＹ-２块茎果聚糖积累量和积累速率的下降幅度都大于ＮＹ-１，ＮＹ-１和ＱＹ-２块茎中果聚糖积累量分别比对照降低了７４．０２％和９３．８１％，块茎果聚糖积累速率降幅达到７７．０８％和９８．４４％。以上结果表明盐胁迫大大降低了两个菊芋品种的生物量，改变了植物体内的糖分分布模式，延缓了果聚糖在体内（尤其是茎）的积累，但南芋一号同青芋二号相比，块茎的产量和果聚糖降幅均较小，说明该品种可用于在部分盐碱地的种植。

牛膝多糖的理化性质研究及结构确证

目的 研究牛膝多糖的理化性质与结构。方法 从中药牛膝中分离得到一小分子量的果聚糖,经凝胶柱色谱分离纯化后,用ESI MS测定其分子量分布。根据甲基化、还原裂解乙酰化及GC MS和NMR分析,确证其结构。结果 单糖组成分析表明牛膝多糖由果糖 葡萄糖(8∶1)组成,其数均分子量为1 400u,含有 4至 21糖;糖链中既含有 1, 2 连接的果糖残基,又含有 2, 6 连接的果糖残基。结论 牛膝多糖为禾本科型果聚糖。

牛膝多糖研究进展

牛膝多糖是从中药牛膝的干燥根里分离得到的果聚糖,相对分子质量小,水溶性好,易被人体吸收,具有广谱的免疫调节活性。牛膝多糖经结构修饰(硫酸酯化,磷酸酯化,羧甲基化,羟甲基化)后得到的一系列的多糖衍生物显示出更高的活性或新的生理活性,具有广阔的药物开发及应用前景。现对牛膝多糖的结构、分离、药理活性、及其衍生化修饰等方面的最新研究进展进行综述。

外源L-色氨酸、果聚糖、酪蛋白对猪粪发酵液粪臭素浓度的影响

为了减少粪臭素在猪体外的污染,本试验旨在研究不同剂量的外源L-色氨酸、果聚糖和酪蛋白对猪粪发酵液中粪臭素及相关合成物浓度的影响。将80头体重为(50.0±0.5)kg的"杜×长×大"三元杂交猪随机分为对照组和试验组,每组4个重复,每重复10头猪,分别饲喂基础饲粮和添加0.2%枯草芽孢杆菌的试验饲粮。6周后,每组随机选取8头猪,采集其新鲜粪便,配制粪水厌氧发酵液用于发酵试验。发酵试验采用3×3正交设计,发酵液中外源L-色氨酸、酪蛋白、果聚糖的添加剂量分别为0、0.05%、0.10%,0、0.125%、0.250%,0、0.75%、1.50%。色谱法测定发酵液中色氨酸、吲哚、吲哚-3-乙酸、粪臭素浓度,荧光标记单链构象多态性及片段长度多态性技术与毛细管电泳技术结合测定空白对照组和空白试验组发酵液中微生物的种群结构。结果表明:1)对照组色氨酸、吲哚、吲哚-3-乙酸、粪臭素浓度显著高于试验组(P<0.05)。2)外源L-色氨酸为发酵液中色氨酸、粪臭素的主要影响因素,酪蛋白为对照组中吲哚的主要影响因素,果聚糖为发酵液中吲哚-3-乙酸的主要影响因素。3)外源L-色氨酸、果聚糖与饲粮中的枯草芽孢杆菌无互作(P>0.05)。4)空白对照组猪粪发酵液优势菌以芽孢杆菌(CWBIB1434)为主,含量为26%;空白试验组发酵液以非解乳链球菌(AF201899)为主,含量为36%。由此得出,降低猪粪中色氨酸的含量及在饲粮中添加枯草芽孢杆菌有助于减少猪粪发酵液中的粪臭素浓度。

转蔗糖:蔗糖-1-果糖基转移酶基因提高烟草的耐旱性

蔗糖:蔗糖-1-果糖基转移酶(sucrose:sucrose 1-fructosyltransferase,1-SST)以蔗糖为底物催化生成蔗果三糖等低聚合度的果聚糖.将从莴苣中克隆的1-SST基因重组到pCAMBIA1300-als中,构建了在CaMV 35S启动子调控下的植物表达载体,利用农杆菌介导的叶盘转化法将1-SST基因导入烟草中,PCR和Southern杂交检测表明获得了转基因植株,RT-PCR结果表明该基因在烟草中正常表达.对T0代转基因烟草进行的耐旱性分析结果表明,干旱胁迫6 d的转基因植株丙二醛含量和电解质渗漏率显著低于未转基因对照,叶片相对含水量下降速度也明显比对照慢.对转基因植株叶片糖分分析表明,转基因烟草植株积累果聚糖,并在干旱胁迫后含量明显增加,而未转基因对照植株不积累果聚糖.在14%PEG溶液中未转基因烟草种子的萌发率仅为转基因烟草种子的一半;在附加200 mmol/L甘露醇的培养基中未转基因烟草种子根的生长明显受到抑制,而转基因烟草根的生长发育正常.以上研究结果表明,转1-SST基因烟草植株耐旱性的提高可能与该基因的表达有关.

内蒙古典型草原羊草和大针茅地下器官中碳水化合物含量的季节性变化

多年生牧草在地下器官中贮藏的碳水化合物是春季萌发生长和刈牧后再生的物质基础。羊草和大针茅是内蒙古典型草原地带性分布的优势种。为了研究草原植物碳水化合物贮藏特性,采用高效液相色谱技术对羊草和大针茅的贮藏性碳水化合物的种类组成及季节变化进行了测定。结果表明,羊草和大针茅地下器官的贮藏性碳水化合物的组分主要包括果聚糖、蔗糖、葡萄糖、果糖、甘露醇和淀粉。其中甘露醇、淀粉和果聚糖在2种牧草地下器官中的含量均居前3位。羊草地下器官碳水化合物的含量显著高于大针茅,其果聚糖和淀粉含量分别是大针茅的3.2和3.6倍。5月20日-10月15日,羊草和大针茅地下器官中的碳水化合物总量、可溶性碳水化合物含量、淀粉含量、甘露醇含量和果聚糖含量均表现出“降低-升高-再降低-再升高”的特征。不同碳水化合物组分冬前积累和春季降解的起止时间也存在较大差异。

转果聚糖合成关键酶基因多年生黑麦草的获得及抗旱性的提高

以多年生黑麦草(品种卡特)胚性愈伤组织为转化受体,利用农杆菌介导的遗传转化方法将冰草果聚糖:果聚糖-1-果糖基转移酶基因(Ac1-FFT)导入黑麦草中,对再生植株喷洒basta溶液和PCR法检测,共获得18个阳性株系,RT-PCR结果表明,该基因在转基因黑麦草中正常表达。转基因黑麦草株系中的可溶性总糖含量和果聚糖含量明显高于对照植株,耐旱性提高,干旱胁迫6d时其相对含水量和叶绿素含量明显高于对照植株,且下降速度慢,但其电解质渗漏率和丙二醛含量显著低于对照植株,复水后很快复原,而对照植株无法恢复,说明转基因植株中由于干旱处理发生的损伤是可逆的,而对照植株中的损伤是非可逆的。以上结果表明,转基因黑麦草中Ac1-FFT的表达及果聚糖合成可能是其耐旱性提高的最重要原因。

去花对菊芋干物质和糖分积累与分配的影响

本研究以菊芋品种青芋二号为试材,在温室进行全生育期栽培试验,研究了青芋二号干物质和糖分积累分配以及去花处理(摘掉花朵)对青芋二号块茎干物质和糖分含量的影响。结果表明,叶片中主要糖分是还原糖,块茎、茎中主要是非还原糖。青芋二号在开花期时总生物量达到最大值,之后地上部生物量减少,地下部由于此时块茎正处于膨大生长阶段,生物量继续增加。去花处理对青芋二号的块茎生物量有着明显的影响,具体表现为块茎数量增多了39.67%,块茎个头变大了59.46%,块茎干重比未去花的增加22.53%。去花植株相同干重下的块茎组织中的总糖含量与未去花植株相比显著降低但还原糖含量没有差异。通过HPLC-ELSD(高效液相色谱-蒸发光检测仪)检测发现去花处理对糖的成分与含量也有影响,蔗果五糖含量增加了32.47%,更高聚合度的果聚糖有增加的趋势,但是其他糖的含量与对照相比差异不大。