东方百合LoABI1基因的克隆及表达分析

该研究以东方百合‘索邦’为材料,采用RT-PCR扩增方法克隆ABI1基因,对其进行了生物信息学分析,并采用实时荧光定量PCR检测其组织表达特性和低温处理过程及定植后的表达特征,以明确ABI1基因的功能特性,为解析百合ABA信号转导途径及其调控低温解除休眠过程的机理奠定基础。结果表明：(1)成功克隆得到东方百合LoABI1基因,其编码序列长度为1 341 bp,共编码446个氨基酸;LoABI1氨基酸序列中含有1个蛋白磷酸酶2C(PP2C)保守结构域。(2)系统进化分析显示,LoABI1蛋白与水稻PP2C家族成员OsPP2C06的同源进化关系最近,且与拟南芥AtABI1聚为一支,同属于PP2C基因家族中的A亚群。(3)亚细胞定位发现,LoABI1蛋白定位于烟草表皮细胞的细胞核和细胞质。(4)qRT-PCR荧光定量分析显示,LoABI1基因在百合茎生根、嫩茎、叶片及各花部组织中均有表达,且在幼嫩组织中表达量较高;LoAB1I基因在冷藏期间的表达量呈先升高后降低的趋势,并于冷藏期第5周达到峰值,但在定植期间持续下降且保持较低水平。(5)经4℃低温处理60 d的百合鳞茎在定植后14～28 d能...     

两步外植体法高频再生麝香百合

采用两步外植体法,即以百合鳞片叶为初始外植体,以从初始外植体上长出的芽为次级外植体,成功建立了麝香百合的高频离体再生系统。对不同的BA浓度及次级外植体的不同部位对再生效果的影响,以及组培苗移栽前低温处理的影响进行了研究。结果表明:不同部位的次级外植体中,以短缩茎切片出芽快、整齐、芽数多且粗壮;以MS附加1.0 mg L-1 BA和0.1 mg L-1 NAA的培养基最适于麝香百合的分化。一个中等大小已脱春化的鳞茎通过两步外植体法能扩繁出54 000株左右的新植株,从鳞片叶开始至开花仅需8个月,而且4!低温处理对开花期的影响不大。     

百合VAL2基因的克隆与表达分析

以东方百合‘索邦’为材料,克隆获得VAL2基因(以下称LoVAL2)。序列分析显示,LoVAL2编码序列长度为2 793 bp,共编码930个氨基酸;预测LoVAL2蛋白具有5个结构域:PHD-L、B3、CW、ZnF-like和EAR结构域。系统进化分析显示,LoVAL2与天门冬亲缘关系较近,与玉米、铁皮石斛等亲缘关系次之,与拟南芥亲缘关系较远。荧光定量表达分析显示,LoVAL2在茎生根、嫩茎、成熟叶、嫩叶及花部组织中均有表达;LoVAL2在雌蕊和雄蕊中的表达于5 mm花蕾中呈现较高水平,随着花蕾发育表达水平下调,之后在雌、雄蕊中的表达水平又分别于20 mm、25 mm花蕾中显著上调,表明LoVAL2基因可能参与雌、雄蕊的早期以及后续发育;亚细胞定位发现LoVAL2在烟草表皮细胞中定位于细胞核,符合转录因子的核定位特征。     

不同激素配比对麝香百合鳞茎芽诱导的影响

采用麝香百合的鳞茎片作为外植体进行组织培养,结果表明,不同激素浓度配比对百合鳞茎芽的诱导效果不同,供试的几个组合中,以MS+6-BA 1.0mg/L+NAA 0.5mg/L效果最好;在继代培养中,以MS+6-BA 2.0mg/L+NAA 0.1mg/L对百合鳞茎的增殖效果最好;在生根培养中,附加活性炭可促进根的生长。     

1,4-β-葡聚糖内切酶基因的克隆及其在麝香百合花粉和花粉管中的特异表达(英文)

利用RT-PCR和RACE在麝香百合(Lilium longiflorum Thunb.)花粉管中克隆到1,4-β-葡聚糖内切酶(Endo-1,4-β-glucanase,EGase)的全长cDNA序列(LlpCell)。序列分析结果表明:该基因编码一个含有490个氨基酸的球状蛋白,并在N端有一个由21个氨基酸构成的信号肽序列。序列比对结果显示,LlpCell和植物分泌型1,4-β-葡聚糖内切酶高度同源(约50％),不含有跨膜结构域和纤维素结合域(CBD)。Northern杂交结果显示,该基因的转录本仅在花粉粒、萌发中的花粉和花粉管生长过程中表达,表达量基本相同。而在百合植株其他组织中均未见表达。这种葡聚糖内切酶的高度特异表达说明LlpCell对花粉萌发和花粉管伸长起着重要作用。     

1,4-β-葡聚糖内切酶基因的克隆及其在麝香百合花粉和花粉管中的特异表达

利用RT-PCR和RACE在麝香百合(Lilium longiflorum Thunb.)花粉管中克隆到1,4-β-葡聚糖内切酶(Endo-1,4-β-glucanase,EGase)的全长cDNA序列(LlpCel1).序列分析结果表明:该基因编码一个含有490个氨基酸的球状蛋白,并在N端有一个由21个氨基酸构成的信号肽序列.序列比对结果显示,LlpCel1和植物分泌型1,4-β-葡聚糖内切酶高度同源(约50%),不含有跨膜结构域和纤维素结合域(CBD).Northern杂交结果显示,该基因的转录本仅在花粉粒、萌发中的花粉和花粉管生长过程中表达,表达量基本相同.而在百合植株其他组织中均未见表达.这种葡聚糖内切酶的高度特异表达说明LlpCel1对花粉萌发和花粉管伸长起着重要作用.     

根癌农杆菌介导麝香百合遗传转化体系的建立

以麝香百合"white elegance"叶片为外植体,通过根癌农杆菌介导法,研究了影响其转化的因素,建立了稳定而高效的麝香百合遗传转化体系。结果表明,以叶片为受体材料,外植体预培养有利于农杆菌的侵染;3 d的共培养,根癌农杆菌OD600值为0.6左右、侵染10 min,能获得较高的转化率;50 mg.L-1的卡那霉素对叶片有很好的筛选效果。抗性植株经PCR检测,初步证明Mn-SOD基因已转入到麝香百合植株中。     

商陆抗病毒蛋白基因导入百合愈伤组织初报

利用美洲商陆抗病毒蛋白(Pokeweed antiviral protein,PAP)具有广谱的抗病毒特性,通过冻融法将含有PAP基因的重组表达载体PBll21转入土壤农杆菌LBA4404中,利用叶盘法在农杆菌的介导下转比麝香百合愈伤组织,通过抗性筛选和PCR输测获得了转PAP基因的百合植株。     

麝香百合抗热性生理生化指标及综合评价初探

以不同高温胁迫(25、38、42、46、50℃)处理麝香百合,依据苗期叶片的相对含水量、脯氨酸含量、可溶性蛋白含量、相对电导率4项指标的变化,对8个基因型进行抗热性综合评价。结果表明,麝香百合不同基因型间的抗热性存在明显差异,8个基因型的抗热性由强至弱顺序:K1-1、K2-7、K1-2、F1、K2-2、Wforest、G、Wfox。     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N-ethylmaleimide inhibition by substrate and Mg²⁺ together suggests sulfhydryl involvement at the active site.     

Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb

Friable callus was obtained from styles and flower pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige and Skoog (MS) media containing either 2 μm dicamba or 2 μm picloram. Cell suspension cultures were established by suspending the callus of L. longiflorum Snow Queen in liquid medium containing 2 μm dicamba. Through a purification process, a fine fast-growing cell suspension was obtained. This suspension was composed of a homogenous population of small dense cells, which tended to organise into embryo like structures (ELS). In liquid culture with the auxin dicamba, the ELS underwent continuous callus formation. When transferred to solidified hormone-free MS medium, the ELS germinated, forming complete plantlets. Histological investigation showed that in the ELS both shoot and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact somatic embryos. Received: 4 April 1997 / Revision received: 13 May 1997 / Accepted: 15 June 1997     

棉花MADS框蛋白基因(GhMADS1)的克隆

作为转录因子,MADS框蛋白基因在植物花器官发育中有着重要的功能。为研究棉花花器官发育的分子机理,以棉花花器官突变体CHV1(cotton homeotic variant)和徐州142正常植株为材料,利用棉花EST数据库资料,通过EST序列整合,从陆地棉徐州142花蕾中克隆出一个MADS框蛋白的编码区段,GenBank登录号为AF538965。该片段(GhMADS1)长713bp,包含一个711bp的开放阅读框,推导的氨基酸序列(236个氨基酸)与葡萄、烟草、矮牵牛、拟南芥和金鱼草等的AGL2组MADS框蛋白有很高的序列相似性。系统进化分析同样将GhMADS1基因归人AGt2组MADS框蛋白。RT-PCR分析显示,该基因在陆地棉的花瓣、雄蕊、胚珠和纤维中表达,特别是在花瓣中表达量最高,而在根、茎、叶等营养器官和棉花同源异型突变体CHV1(所有花器官均变为苞叶状叶性器官)的变异花蕾中不表达。这些结果说明GhMADS1基因可能在棉花花器官发育中有着重要的功能。     

Phenolic glycosides from Lilium longiflorum

Three bitter principles were isolated from the bulb scales of Lilium longiflorum and identified as 3,6′-diferuloylsucrose, 4-acetyl-3,6′-diferu     

A Calcium-Activated Phytase from Pollen of Lilium longiflorum

A phytase was isolated and partially purified from the pollen of Lilium longiflorum Thumb. Optimum activity was at pH 8.0. The phytase was activated by Ca²⁺ and Sr²⁺ but not by the other divalent cations tested. Activity was inhibited by ethylenediaminetetraacetate. The phytase had a temperature optimum of 55 to 60°C and an activation energy of about 12,700 calories/mole. Extraction of L. longiflorum pollen with 0.1% Triton X-100 increased recovery of the phytase by nearly 4-fold. The phytase had a molecular weight of about 88,000 as determined by gel filtration chromatography and a K_m value of 7.2 micromolar for phytic acid in the presence of Ca²⁺.     

Partial purification and characterization of phytases from pollen of lily (Lilium longiflorum Thunb.)

Summary Two phytases from lily pollen (Lilium longiflorum Thunb.) were partially purified and characterized. The first (pH optimum 5.0) was purified 40-fold from ungerminated pollen. The second (pH optimum 6.5) appeared during germination and was purified 68-fold from pollen germinated 2 h. Molecular weight of the first was 72 kD, and the second was 36 kD as determined by gel filtration. Both were active against phosphate esters other than phytate, although purification of the first reduced its activity against AMP and myo-inositol 2-P to 10% of activity against phytate. Phytase from germinated pollen (but not ungerminated) was inhibited by the sulfhydryl agent parahydroxy mercuribenzoate; P _i inhibited phytase from ungerminated but not germinated pollen. Such different catalytic and physical properties may reflect different biochemical functions.Abbreviations HPLC High performance liquid chromatography - DEAE diethyl aminoethyl - P _i orthophosphate - PP _i pyrophosphate - p-NPP para-nitrophenyl phosphate - pNP para-nitrophenol - MI myo-inositol - MI 2-P myo-inositol 2-P - MI penta P myo-inositol pentakisphosphate - PHMB para-hydroxy mercuribenzoate - PMSF phenyl methyl sulfonyl fluoride - AMP adenosine monophosphate - GMP guanosine monophosphate - EGTA ethylene glycol-bis (-aminoethyl ether) N, N, N, N-tetraacetic acid     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style     

岷江百合类萌发素蛋白基因LrGLP2的克隆及表达特性分析

类萌发素蛋白是植物中普遍存在的一类可溶性糖蛋白,在植物抗逆胁迫中起着重要作用。依据岷江百合编码GLP的EST序列设计引物,采用快速扩增cDNA末端技术,从岷江百合犯ilium regale Wilson）克隆得到一个新的GLＰt基因的全长eDNA序列,命名为LrGLP2。LrGLP2全长cDNA为921bp,含有654bp的开放阅读框,49bp5’非编码区以及218bp3’UTR,编码217个氨基酸的蛋白质。LrGLP2编码蛋白质与已知植物GLPs家族成员间的同源性和聚类分析表明LrGLP2与来源于水稻（Oryza sativa）、节节麦似egilopstauschii）、葡萄（Vitis vinifera）中的GLPs具有较高的相似性。qRT-PCR分析显示,LrGLP2在岷江百合正常生长发育的根中有一定量的表达,而在茎和叶中几乎检测不到表达量。水杨酸、茉莉酸以及H202处理均不同程度抑制LrGLP2的转录水平,但乙烯处理能明显诱导LrGLP2的表达。此外,岷江百合接种尖孢镰刀菌（Fusari—umoxysporumfsp．tiliO后,LrGLP2在接种后2h表达迅速上调,12h表达量急剧上升,至24h表达量达到最大值,之后表达量下降,可见￡rGLP2参与岷江百合对尖孢镰刀菌的防卫反应。