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。

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。

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.

Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

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.

百合病毒的多重RT-PCR检测技术

根据百合(Lilium brownii var.viridulum)常见的黄瓜花叶病毒(CMV)、百合斑驳病毒(LMoV)、无症病毒(LSV)引物序列,设计2组特异引物,以18S rRNA为内参基因,采用多重RT-PCR,2组引物均能扩增得到LMoV和LSV预期大小一致的片段,未检测到CMV,检测结果不受病株取材部位的影响。该方法的建立为百合脱毒苗的规模化生产提供了高效检测技术。

补光及施肥对东方百合‘玛丽’切花生长和品质的影响

以东方百合品种‘玛丽’(Mero star)为材料,研究了补光和施肥对其生长发育和开花的影响。结果表明:补光促进了开花,能提早20 d左右,使切花品质提高,花败育减少,促进了花色鲜艳。而施肥对开花早晚无明显影响,但在补光条件下随着施肥水平的提高品质提高。

麝香百合花粉发芽适宜培养基及其贮藏研究

采用不同培养基对麝香百合(Liliumlongiflorum)花粉的发芽率进行了对比研究。结果表明,在空白培养基上其花粉发芽率仅为4.7%,而在加有蔗糖、锌、硼的培养基上,麝香百合花粉的发芽率有大幅度的提高,其中以培养基10%蔗糖+100mg.L-1硼+0.02mg.L-1锌为最佳,花粉发芽率可达79.7%;对不同贮藏条件下麝香百合花粉的活力测定结果表明,在低温干燥条件下贮藏能较长时间地保存其生活力。

百合离体抗盐变异体的筛选及生理特性研究

用甲基磺酸乙酯(EMS)对百合离体叶片进行了处理。结果表明,随着处理浓度和处理时间的增加,叶片的分化率和存活率均下降,并筛选出其半致死浓度(0.6%)和处理时间(3 h)。利用筛选的EMS浓度和处理时间对离体叶片进行处理,在1%盐浓度胁迫下进行抗盐性突变体筛选,得到抗盐性诱变植株。经鉴定,其超氧化物歧化酶(SOD)、过氧化物酶(POD)活性增强,丙二醛(MDA)含量减少,细胞膜透性减小,可溶性蛋白含量增加,表明筛选的百合突变体比对照具有更强的抗盐性。

GA_3、IBA和6-BA对香水百合开花特性及内源激素的影响

采用正交设计研究了6-苄氨基嘌呤(6-BA)、赤霉素(GA3)、吲哚丁酸(IBA)3种植物生长调节剂对香水百合(Lilium casa blanca)开花特性及内源激素的影响。结果表明,以150 mg/L GA3+40 mg/L IBA+60 mg/L 6-BA和40 min的浸球处理组合最佳,可使香水百合的花期提前,而且改善了花的品质,蕾长增加,花径增大。植物生长调节剂与香水百合开花性状的相关分析表明,外源6-BA对开花性状起主要作用。内源GA3和IAA含量的变化趋势一致,从现蕾期到盛花期呈下降趋势,花衰败后上升;内源ABA含量随着花的形成含量逐渐升高;内源ZT含量在盛花期下降,花衰败后再次上升。内源激素的动态变化影响了香水百合的开花特性,不同时期内源激素与开花特性的相关性不同。

秦巴山区百合种质资源及开发利用

秦巴山区是中国野生百合（Lilium L.）集中分布地区之一。以梳理该地区的百合种质资源为基础,对秦巴山区野生百合资源的分布、遗传多样性以及目前的开发利用现状进行了概述,以期为开发和保护秦巴山区百合资源提供理论依据。

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.

宜兴百合鳞茎不定芽的诱导及增殖

以宜兴百合（Lilium lancifalium Thunb.）鳞片为外植体诱导小鳞茎的形成,并通过正交设计增殖鳞茎不定芽.结果在MS＋1.0 mg/L 6-BA＋ 0.1 mg/L NAA培养基上,鳞片以直接发生方式形成了鳞茎不定芽,诱导率为100％,平均不定芽数为5.5.6-BA浓度在0.4～1.2 mg/L范围内,鳞茎不定芽的增殖率随6-BA浓度的升高而增大,在MS＋1.2 mg/L 6-BA ＋0.2 mg/L IAA ＋0.1 mg/L NAA培养基上,不定芽的增殖系数为3.83,不定芽在1/2MS＋1.0 mg/L NAA培养基上生根良好.

淡黄花百合花期生物量分配

[目的]研究淡黄花百合(Lilium sulphureum Bakerapud Hook.f.)花期生物量分配,以期为黄花百合的田间管理和合理采摘提供基础资料。[方法]对贵州省务川县境内野生淡黄花百合花期的不同构件进行了称量,用SPSS软件进行相关性检验及回归分析。[结果]淡黄花百合在花期各构件的生物量分配顺序为鳞片>地上茎>叶>花>地下茎>茎生根>鳞茎盘或基盘根。作为百合粉原料的鳞片生物量分配在鲜重时(33.4%)和干重时(41.2%)均为最大。鳞片干重生物量与植株基径、株高及其他构件干重生物量均呈极显著正相关(P<0.01)。鳞片干重生物量分配与地上茎和地下茎的干重生物量分配均呈极显著负相关(P<0.01),与植株生殖构件(花)的干重生物量分配呈显著负相关(0.01<P<0.05)。[结论]淡黄花百合植株在养分分配及物质积累与植株的生长上存在着内在协调性联系。在人工栽培过程中应注意控制生殖构件和茎对鳞片生长的影响。

细叶百合鳞茎多酚类物质组成及其抗氧化活性

对野生细叶百合(Lilium pumilum DC.)鳞茎多酚类物质组成及其抗氧化能力进行研究,为野生百合资源的合理利用提供科学依据。以宁夏罗山野生细叶百合鳞茎为材料,比较经D-101大孔树脂纯化处理前后细叶百合总多酚含量的变化,使用高效液相色谱法检测分析细叶百合鳞茎内多酚类物质的组成与含量,采用清除DPPH、超氧根离子和羟基自由基3种方法对百合多酚提取物的总抗氧化活性进行评价。结果表明,经D-101大孔树脂纯化后其多酚含量有所降低,但回收率高达92.51%;经高效液相检测分析,11种单体酚化合物在细叶百合鳞茎中被检出,其中杨梅酮和表儿茶素的含量相对较高,分别达到了4.48 mg/100 g和2.91 mg/100 g;细叶百合多酚提取物对DPPH、超氧根离子和羟自由基均具有良好的清除能力,可作为天然抗氧化剂被开发利用。

无银保鲜剂对卷丹百合切花的保鲜效果

为了进一步延长切花卷丹百合(Lilium brownii var.viridulum Baker)的瓶插寿命,提高观赏价值,分析了不同保鲜剂对卷丹百合的保鲜效果,以3%蔗糖+150 mg/L 8-HQ作为基础液,分别添加1.5mmol/L STS与100 mg/L Al2(SO4)3,测量其瓶插寿命、鲜重变化率、水分平衡值以及花瓣中MDA的含量。结果表明,保鲜剂B[3%蔗糖+150 mg/L 8-HQ+100 mg/L Al2(SO4)3]的保鲜效果较好,其瓶插寿命延长了5.2 d,鲜重变化率最高,能够很好地促进鲜切花的吸水量,并且花瓣中MDA的含量较低,能够有效地维持细胞膜结构的稳定性,使切花百合的观赏效果达到最佳。

神农架自然保护区宜昌百合染色体核型分析

[目的]研究神农架自然保护区宜昌百合染色体核型,为研究宜昌百合遗传多样性、起源及系统演化提供一定的细胞学依据。[方法]采用常规制片法对神农架保护区的宜昌百合进行染色体核型分析。[结果]神农架保护区宜昌百合染色体数目为2n=2x=24,染色体基数x=12,染色体核型公式为K(2n)=2x=24=4m(2SAT)+2sm+8st+10 t,主要由端部和近端部着丝点染色体组成。染色体相对长度组成为2n=24=4L+4M2+14M1+2S,核型不对称系数为79.66%,属于"3B"型。[结论]神农架宜昌百合在百合属植物系统演化上处于比较进化的类型。

百合枯萎病病原菌分离鉴定及药剂室内筛选

采用组织分离法对枯死的百合(Lilium brownii var.viridulum Baker)鳞茎进行病原分离,根据分离物的培养性状、颜色、孢子大小,结合显微镜检测和分子生物学ITS序列测定,该菌株被鉴定为尖孢镰刀菌(Fusarium oxysporum)。对菌株进行柯赫氏法则验证,证明尖孢镰刀菌是导致百合枯萎病的病原菌。选择15种杀菌剂对该病原菌进行室内毒力测定。结果表明,广清、绿群、洛菌腈、氟硅唑抑菌效果明显强于其他药剂。