WRKY11 up-regulated dirigent expression to enhance lignin/lignans accumulation in Lilium regale Wilson during response to Fusarium wilt

Lilium are highly economically valuable ornamental plants that are susceptible to Fusarium wilt caused by Fusarium oxysporum.Lilium regale Wilson,a wild lily native to China,is highly resistant to F.oxysporum.In this study,a WRKY transcription factor,WRKY11,was isolated from L.regale,and its function during the interaction between L.regale and F.oxysporum was characterized.The ectopic expression of LrWRKY11 in tobacco increased the resistance to F oxysporum,moreover,the transcriptome sequencing and UHPLC-MS/MS analysis indicated that the methyl salicylate and methyl jasmonate levels rose in LrWRKY11 transgenic tobacco,meanwhile,the expression of lignin/lignans biosynthesis-related genes including a dirigent(DiR)was up-regulated.The lignin/lignans contents in LrWRKY11-transgenic tobacco also significantly increased compared with the wild-type tobacco.In addition,the resistance of L.regale scales in which LrWRKY11 expression was silenced by RNAi evidently decreased,and additionally,the expression of lignin/lignans biosynthesis-related genes including LrDIR1 was significantly suppressed.Therefore,LrDIR1 and its promoter(PLrDIR1)sequence containing the W-box element were isolated from L.regale.The interaction assay indicated that LrWRKY11 specifically bound to the W-box element in PLrDIR1 and activated LrDIR1 expression.Additionally,β-glucuronidase activity in the transgenic tobacco co-expressing LrWRKY11/PLrDIR1-β-glucuronidase was higher than that in transgenic tobacco expressing PLrDIR1-β-glucuronidase alone.Furthermore,the ectopic expression of LrDIR1 in tobacco enhanced the resistance to F.oxysporum and increased the lignin/lignans accumulation.In brief,this study revealed that LrWRKY11 positively regulated L.regale resistance to F.oxysporum through interaction with salicylic acid/jasmonic acid signaling pathways and modulating LrDIR1 expression to accumulate lignin/lignans.

R2R3-type Lo MYB21 affects jasmonate-regulated development and dehiscence of anthers in lily(Lilium oriental hybrids)

Lilies are widely cultivated for cut flowers,but their large anthers carry a considerable amount of colored pollen that is dispersed easily.Studying the molecular mechanism of anther development and dehiscence could help solve this problem.LoMYB21,encoding a putative R2R3v-myb avian myeloblastosis viral oncogene homolog(MYB)transcription factor,was identified from oriental lilies(Lilium‘Siberia’).Real-time quantitative PCR analysis showed that LoMYB21 was mainly expressed in the anther,filament and stigma and had high expression during the late stages of lily anther development.LoMYB21 had transactivation activity and was located in the nucleus through yeast one-hybrid assays and transient expression in Nicotiana benthamiana.Suppression of LoMYB21 by virus-induced gene silencing(VIGS)in Lilium‘Siberia’led to anther indehiscence and reduced the expression of genes related to Jasmonate acid(JA)biosynthesis and signal transduction.Induction of LoMYB21 in DEX::LoMYB21 transgenic Arabidopsis caused procumbent inflorescences that became infertile,accompanied by higher expression of JA biosynthetic and signaling genes.These results demonstrated that JA content and signaling were abnormal in silenced lily and transgenic LoMYB21 Arabidopsis,which affected anther development.Our study indicated that LoMYB21 could regulate lily anther dehiscence through JA biosynthesis and signaling during the late stages of anther development.

Monitoring Xylem Transport in the Stem of Lilium lancifolium Using Fluorescent Dye 5(6)-Carboxyfluorescein Diacetate

The xylem undergoes physiological changes in response to various environmental conditions during the process of plant growth.To understand these physiological changes,it is extremely important to observe the transport of xylem.In this study,the distribution and structure of vascular bundle in Lilium lancifolium were observed using the method of semithin section.Methods for introducing a fluorescent tracer into the xylem of the stems were evaluated.Then,the transport rule of 5(6)-Carboxyfluorescein diacetate(CFDA)in the xylem of the stem of L.lancifolium was studied by fluorescence dye in live cells tracer technology.The results showed that the vascular bundles of L.lancifolium were scattered in the basic tissue,the peripheral vascular bundles were smaller and densely distributed,and the closer to the center,the larger the volume of vascular bundles and the more sparsely distributed.The vascular bundles of L.lancifolium are limited external tenacity vascular bundles,which are composed of phloem and xylem.The most suitable method for CFDA labeling the xylem of isolated stem segments of L.lancifolium was solution soaking for 24 h.The running speed of CF in the isolated stem was 0.3 cm/h,which was consistent with the running speed of the material in the field.CF could be transported between the xylem and parenchyma cells,indicating that the material transport in the xylem could be through the symplastic pathway.The above results laid a foundation for the study of the xylem transport mechanism and the xylem pathogen disease of lily.

Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium‘Siberia’

Terpenoids are the main components contributing to the fragrance of Lilium‘Siberia’,and LiTPS2 plays a critical role in the biosynthesis of monoterpenoids.Although the major terpene synthases in Lilium‘Siberia’have been identified,how these TPS genes are transcriptionally regulated remains elusive in this distinguished flower.This study aimed to identify transcription factors that regulate the terpene synthesis in Lilium,and disclose the related underlying transcriptional regulation mechanism.In this study,we identified three R2R3-MYB TFs—LiMYB1,LiMYB305 and LiMYB330,which were involved in regulating the biosynthesis of terpenes in Lilium‘Siberia’.Quantitative real-time PCR showed spatial and temporal expression patterns consistent with the emission patterns of terpene compounds.When LiMYB1,LiMYB305 and LiMYB330were overexpressed in flowers,the release of some main monoterpenes,such as linalool and ocimene,as well as the expression of TPS genes,especially for LiTPS2,were enhanced.A virus-induced gene silencing(VIGS)assay showed that silencing these three LiMYBs decreased the level of monoterpenes by down-regulating the expression of the TPS genes.The yeast one-hybrid and transient expression assays indicated that all three LiMYBs could bind to and activate the promoter of LiTPS2.Moreover,the yeast two-hybrid assay verified that LiMYB1 could interact with LiMYB308 and LiMYB330,indicating their synergistic roles in the regulation of floral terpene biosynthesis.In general,these results indicated that LiMYB1,LiMYB305,and LiMYB330 might play essential roles in terpene biosynthesis in Lilium and would provide a new perspective for the transcriptional regulation of volatile terpenes in flowers.

兰州百合鳞茎花青素合成相关MYB与bHLH转录因子的筛选分析

百合鳞茎在采后贮藏过程中,由于花青素的积累导致鳞茎变紫红色,进而影响其价值。该文基于兰州百合鳞茎转录组数据,利用生物信息学技术分析花青素相关转录因子,共分析了50个MYB转录因子与73个bHLH转录因子,包括序列的比对、理化性质分析、亚细胞定位、系统进化关系以及保守结构域预测。MYB分类结果显示,50个MYB转录因子中有2个属于1R-MYB类,44个属于R2R3-MYB类,3个属于3R-MYB类,1个属于4R-MYB类。50个MYB不稳定蛋白均为亲水性蛋白,亚细胞定位表明48个MYB定位于细胞核。73个bHLH均为不稳定蛋白,且72个都为亲水性,亚细胞定位显示58个bHLH定位于细胞核。通过与模式植物拟南芥MYB和bHLH转录因子构建进化树分析、保守结构域预测、基因表达量分析以及转录因子与结构基因的相关性分析,初步筛选出3个MYB基因和1个bHLH基因可促进兰州百合鳞茎花青素合成,为进一步深入研究兰州百合鳞茎花青素调控基因提供了理论支持。

龙牙百合褐斑病病原的鉴定、生物学特性及室内药剂筛选

为明确龙牙百合褐斑病病原,采集龙牙百合褐斑病典型症状的叶片,进行病原物分离、纯化和致病性测定,结合病原菌形态学观察及其rDNA-ITS、gapdh、tef1、RPB2和Alt a1等基因的序列分析,确定引起龙牙百合褐斑病的病原菌为枝状链格孢Alternaria arborescens。采用菌丝生长速率法,测定了光照、温度、pH值、碳源、氮源和不同培养基等处理对该病原菌生长的影响,并测定了病原菌对7种杀菌剂的敏感性。结果表明,最适合菌丝生长的条件为温度25℃,pH值5.0,碳源为蔗糖,氮源为牛肉浸膏,培养基为马铃薯葡萄糖琼脂(PDA)培养基;菌落致死温度为50℃。在7种参试药剂中,木霉菌抑制作用最强,其半数效应浓度(EC_(50))和90%效应浓度(EC_(90))值最低,分别是8.758×10^(-5)和0.198 mg·L^(-1);噁唑菌酮代森锰锌的毒力最低,EC_(50)和EC_(90)值分别为13.713和816.937 mg·L^(-1)。本研究结果为龙牙百合褐斑病菌的精准防控提供了理论依据。

百合育种技术研究进展

百合(Lilium spp.)是多年生球根草本植物,包括观赏、食用和药用百合,具有较高的经济价值。百合遗传背景复杂、杂合度高、远缘杂交不亲和,且传统杂交育种周期长、育种精度低,难以快速高效地培育目标品种。目前,包括现代杂交育种、诱变育种、倍性育种、体细胞杂交育种和基因工程育种在内的现代育种技术均已在百合中应用。本文总结了百合杂交育种中远缘杂交不亲和的克服方法,诱变育种中的诱变条件,倍性育种中多倍体育种和单倍体育种主要采取的技术手段,体细胞杂交育种原生质体分离纯化和杂交的条件以及基因工程育种中百合遗传转化体系和基因编辑体系的研究进展。在此基础上,列举了不同百合育种方法和技术的研究案例,分析了不同育种技术面临的问题,并展望了百合育种技术的进一步发展和应用前景,旨在为未来百合育种技术及其应用的研究提供有价值的参考,并为更多百合新品种的创制提供依据。

卷丹百合黄酮提取物抗氧化活性研究

该实验旨在研究卷丹百合黄酮提取物抗氧化活性。以化学法、秀丽隐杆线虫模型为评价方法,用自由基清除率、抗氧化酶活力及线虫寿命等为指标,探究卷丹百合黄酮化合物的抗氧化能力。结果表明,卷丹百合黄酮提取物在料液比为1:10(g:mL)时,抗氧化活性达到最大值,其中DPPH自由基清除率为83.46%,ABTS阳离子自由基清除率为73.26%。卷丹百合黄酮提取物能显著提高线虫体内抗氧化酶活力和谷胱甘肽含量,提高线虫抗氧化能力,起到延长线虫寿命的作用。经过卷丹百合黄酮提取物处理的线虫在氧化应激条件下平均存活时间能达到10 h。卷丹百合黄酮提取物具有良好的抗氧化活性,具备开发食品抗氧化剂或功能食品的潜力。

基于UHPLC-QTOF-MS的兰州百合植物化学成分鉴定

为探究地理标志产品兰州百合(Lilium davidii var.unicolor)的植物化学成分,以甘肃省五个主要产区(兰州市榆中县、兰州市七里河区、定西市临洮县、定西市渭源县、临夏市永靖县)的兰州百合鳞茎为研究对象,基于超高效液相色谱串联四极杆飞行时间质谱法的非靶向代谢组学技术鉴定其植物化学成分。结果表明,在兰州百合鳞茎中初步鉴定出62种植物化学成分,包含谷胱甘肽等22种氨基酸、多肽及其衍生物,溶血磷脂酰胆碱等9种油脂和磷脂类,鼠李糖等7种糖类,阿魏酸、咖啡酸等7种酚酸类,山奈酚等4种黄酮类,核黄素、维生素C等4种天然维生素,阿魏酰腐胺等3种酚胺类,大丁苷等2种香豆素类,葫芦巴碱等2种生物碱,萜类组分松香酸和三萜皂苷类组分刺五加苷E。本研究丰富了兰州百合植物化学成分的现有数据,为兰州百合的研究与利用提供了科学依据。

The Study on Induction and Proliferation of Tube Bulbs in Lilium brownii

With different parts of bulb scale as explants, the proliferation method of Guizhou Lilium brownii were studied with 3% sodium hypochlorite and MS medium with different concentrations of hormones. The results show that it is feasible to disinfect the bulbs of Lilium brownii with 3% sodium hypoehlorite, moreover, the sodium hypochlorite is very cheap and harmless to researchers, experimental materials and environment. MS ＋ NAA 0.3 mg/L ＋ 6-BA 1.5 mg/L is optimum for the induction of bulbs and, the basal part of Lilium brownie is the optimum explants. After culture for 25 d on the same medium, the tube bulbs could be obtained with the characteristics of high propagation coefficient, strong and new roots. The survival rate is over 90% for transplantation of tube bulbs with diameter between 1-2cm. The method developed in the present study can proliferate abundant Lilium brownii seedling in short time.

百合属(Lilium)几种植物花粉形态的观察

应用光学显微镜和扫描电子显微镜对百合属(Lilium)植物13个种花粉的形态、表面纹饰、萌发沟等性状的观察结果表明,百合属植物花粉皆为椭球形,具单萌发沟,网状纹饰,而花粉大小、网脊宽度、网眼直径等性状存在差异。在供试的13个种中,麝香百合系品种罗瑞拉(Longiflorum hybrids cv.Lorina)的花粉最大,有斑百合(L.concolor var.buschianum Baker)的花粉最小。花粉形态特征和超微结构对百合属植物的分类有一定价值。

Study on the Integration of T-DNA into Lilium longiflorum

[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobacterium mediated method. [ Result] The best infection effect appeared under the OD600 value of Agrobacterium within 0.6 -0.8, the addition of 250 mg/L AS could increase the transformation efficiency. The optimal concentration of G418 for screening is 50 mg/L. Some putative transgenic plants of Lilium longiflorum with resistance to G418 showed positive in PCR, preliminarily proving that T-DNA gene had integrated into the genome of lily. [ Conclusion] The study may lay a foundation for breeding excellent lily varieties through TDNA integration technique.

东方百合(Lilium oritential)组织培养研究

以东方百合"索邦"为研究对象,对东方百合组织培养技术进行研究。结果表明:启始培养所用材料的基部诱导率明显高于上部,鳞茎分化芽的数量明显多于鳞片;MS+0.1 mg.L-1BA+0.1 mg.L-1NAA为最佳鳞茎诱导培养基;MS+0.1 mg.L-1BA+0.3 mg.L-1NAA为最佳鳞茎增殖培养基;用1/2MS+0.1 mg.L-1IBA培养基进行生根培养,取得良好的效果。

褪黑素诱导西伯利亚百合的转录组分析及LoERF96基因克隆

【目的】研究西伯利亚百合萌发阶段的分子调控机制,筛选响应褪黑素、促进百合鳞茎萌发的潜在乙烯响应因子(ethylene response factor,ERF)。【方法】对褪黑素处理下的西伯利亚百合小鳞茎关键时期样本进行转录组测序分析,并对差异表达基因进行功能注释,筛选响应褪黑素、促进百合鳞茎萌发的ERF潜在基因。对筛选的关键基因LoERF96进行克隆和生物信息学分析,通过实时荧光定量分析小鳞茎4个不同生长发育时期中LoERF96基因的表达量。【结果】根据转录组数据筛选得到780个差异表达基因,其中上调表达基因392个,下调表达基因388个。GO富集分析结果显示:富集的差异基因主要生物学过程包括代谢过程、细胞过程、生物调节、刺激响应、信号、生殖过程等。KEGG代谢途径分析结果显示:差异基因的代谢途径包括植物激素信号转导、植物MAPK信号途径、苯丙烷生物合成、淀粉和蔗糖代谢、光合作用、碳代谢、内质网中的蛋白质加工等。通过同源克隆得到LoERF96基因,该基因序列全长435 bp,编码144个氨基酸,有1个AP2保守结构域,蛋白相对分子质量约为16.32 ku,理论等电点为4.94,是没有跨膜结构的亲水性蛋白,预测亚细胞定位于细胞核中。RT-qPCR表达结果显示:在百合4个不同生长发育过程中,LoERF96基因的相对表达量总体呈先上升后下降的趋势。【结论】西伯利亚百合中的乙烯响应因子LoERF96受褪黑素诱导表达,这一结果为探究百合AP2/ERF转录因子萌发阶段的生物学功能提供了理论基础和参考。

百合花香物质合成与调控机制研究进展

百合(Lilium spp.)原产于我国,在我国花卉产业中有着重要而特殊的地位。花香是其观赏性状的重要标签,已有研究结果表明,不同香型的百合主要花香成分差异较大,浓香型与淡香型百合的差异主要集中在萜烯类物质的不同,而采样时期、部位、环境、激素等均会导致花香成分变化。目前百合花香物质合成通路研究集中在萜烯合成通路上,多为萜烯合酶的功能研究及上游调控网络解析,而其他花香成分代谢途径基因的功能解析及分子调控机制的研究仍存在许多未解之谜。由于对百合花香合成及调控机制的解析不够全面与深入,难以支撑花香的精准改造,导致百合花香育种进程缓慢。深入挖掘与利用百合花香基因、完善相关代谢途径及调控网络可能会是百合花香的下一步研究重点。本文对百合花香的前期研究进行了回顾和总结,并对后期的研究方向提出展望,以期为后续百合花香分子调控机制研究提供参考,对定向培育香气怡人的百合新品种提供借鉴。

催花保鲜剂对百合(Lilium)绿蕾催花保鲜生理的研究

探讨了百合切花绿蕾经两种催花保鲜剂处理后的效果 ,结果表明 ,催花保鲜剂能促进绿蕾提前开花 ,同时对开花后的百合切花有延缓衰老的作用 ,减少了切花在瓶插期间叶绿素与蛋白质含量的降解 ,提高了切花的品质和商品价值。处理 增加了开放率和花蕾长度 ,处理 增加了花瓣长和花径大小 ,同时在试验过程中揭示了百合切花经催花剂处理后的水分生理变化规律 ,筛选出最佳的催花保鲜剂为处理 。

百合鳞茎营养素与植物化学成分研究进展

百合是多年生草本植物,兼具观赏、食用和药用价值,其可食用或药用部位一般为鳞茎。近年来,百合鳞茎作为一种特色蔬菜,受到消费者青睐。本文重点从不同品种视角对百合鳞茎进行了分析,首先归纳了碳水化合物(包括活性多糖)、蛋白质及氨基酸、脂类、矿物质和维生素等营养素的详细构成,进一步总结了其甾体皂苷(包括螺甾烷醇、异螺甾烷醇、变形螺甾烷醇、呋甾烷醇类)、酚酸类(包括单聚酚酸、酚酸甘油酯和苷类)、黄酮类(包括黄酮醇、黄烷醇、查尔酮、黄烷酮、黄烷酮醇、花色苷类等)和生物碱类(包括异喹啉、吡咯啉、哌啶和甾体糖苷类)等已知植物化学成分的构成及其生物活性,最后对百合在保健食品中的应用进行概括,以期为百合研究及非药用加工利用提供理论依据和研究思路。

兰州百合响应自毒胁迫的代谢组学分析

为探究兰州百合(Lilium davidii var.unicolor)响应自毒胁迫的代谢机制,以1年生(Lily1)、2年生(Lily2)和3年生(Lily3)兰州百合为研究对象,采用气相色谱-质谱(GC-MS)技术分析了不同连作年限兰州百合的代谢组学变化,在其提取物中共检测和鉴定出124种代谢物。利用R软件对样品进行归一化,通过正交偏最小二乘判别分析(OPLS-DA)寻找差异代谢物,并筛选相关代谢物途径进行联合分析。结果表明:Lily1与Lily2的代谢组间存在明显差异,共有16种呈上调的差异代谢物(脂质类、黄酮类、糖类及酚类),且均上调;显著富集至酪氨酸代谢、丙酸代谢和类黄酮生物合成代谢通路。其中脂质类、黄酮类和糖类物质含量的提升表明在兰州百合连作的第2年,自身通过积极合成脂质类、黄酮类和糖类物质来响应自毒胁迫。Lily1与Lily3的代谢组间共有21种差异代谢物,包括1种上调代谢物(3-α-甘露二糖)和20种下调代谢物(胺类、酸类和脂质类);显著富集至5条代谢通路,分别为托烷、哌啶和吡啶生物碱的生物合成、磷酸盐和次磷酸盐的代谢、赖氨酸降解、谷胱甘肽代谢和D-氨基酸代谢。Lily2与Lily3组间同样有21种差异代谢物,唯一上调代谢物也为3-α-甘露二糖。这表明,在兰州百合连作的第3年,糖类物质作为直接参与维持细胞膜稳定性的碳水化合物,积极响应自毒胁迫。20种下调代谢物分别为胺类、酸类和脂质类,仅显著富集至丙酸代谢和类黄酮生物合成代谢通路。

兰州百合多倍体离体诱导及其分子细胞鉴定

兰州百合是我国最重要的可食用甜百合。为培育优质的百合新种质,用不同浓度的秋水仙素离体诱变兰州百合小鳞茎,通过不定芽诱导获得变异幼苗。利用染色体倍性分析、幼苗早期形态观察及ISSR分子标记对其进行鉴定。结果表明,用0.15%秋水仙素处理小鳞茎48 h诱导效果最好,变异率为58.06%。选择2个形态变异且生长较快的幼苗进行流式细胞仪分析和根尖压片计数,发现其中1个变异株系含36条染色体的细胞数比例达到了80%,接近纯合三倍体。形态学观察显示,2个变异幼苗的叶片、鳞茎及根系与对照相比存在显著差异,且气孔保卫细胞大于对照,气孔密度降低。ISSR标记检测表明,2个诱变株系与对照相比均发生遗传变异,变异率分别为19.64%和20.91%。研究结果可为获得兰州百合三倍体新种质及秋水仙素诱变育种提供重要参考依据。

卷丹百合LlARF12基因的克隆、表达及功能分析

【目的】探究卷丹百合珠芽发生前细胞和组织学层面的变化,讨论卷丹百合珠芽发生的内在机制。克隆生长素响应因子基因LlARF12,为解析LlARF12基因在卷丹百合珠芽发生过程中的作用机制提供参考。【方法】利用石蜡切片的方法对卷丹百合珠芽器官形成前的叶腋部位进行了组织学观测,从卷丹珠芽形成过程的转录组中筛选得到参与珠芽发生的关键基因LlARF12,克隆得到其编码区(CDS)序列,利用生物信息学软件对其进行分析预测,通过qRT-PCR对不同时期、不同种与品种的百合组织中的LlARF12基因进行表达模式分析,并通过VIGS技术对卷丹种球进行LlARF12基因沉默,从而验证其基因功能。【结果】克隆获得LlARF12基因长度为2346 bp,编码781个氨基酸;qRT-PCR结果显示,LlARF12在珠芽发生的小白点时期表达量显著低于未发生珠芽时期、在S1时期植株茎秆上部表达量显著低于茎秆下部、在能够自然发生珠芽的百合品种中表达量显著低于其他百合品种;沉默LlARF12基因导致卷丹珠芽发生时间提前,且发生的珠芽数量增多。【结论】在卷丹百合珠芽发生的S1时期之前,腋生分生组织已经开始启动,此处靠近表皮的薄壁细胞层数增加、细胞核堆积,并且逐渐产生成熟的维管组织。LlARF12在卷丹百合不能发生珠芽的组织部位和时期的表达量普遍更高,推测其在珠芽发生过程中可能发挥转录抑制作用,通过VIGS技术进行的基因功能验证证明了LlARF12对珠芽发生的抑制作用。