6个香雪兰品种的染色体核型及聚类分析

以香雪兰（Freesia hybrida）6个品种的根尖为研究材料进行核型分析以揭示其核型特征,进一步通过聚类分析品种间的相似性并探讨可能的亲缘关系。结果表明：香雪兰6个品种均为四倍体,染色体数目都为44。6个品种的核型公式不同：‘Gold River’为2n=4x=44=33m+11sm,‘White River’为2n=4x=44=1M+34m+9sm,‘Castor’为2n=4x=44=35m+9sm,‘上农红台阁’为2n=4x=44=32m+12sm,‘Pink Passion’为2n=4x=44=38m+6sm,‘Red River’为2n=4x=44=37m+7sm。核型参数因品种而异,其中,‘Castor’在相对长度范围和臂比范围上均为最广,‘上农红台阁’核不对称系数最大。6个品种核型不对称系数为57.00%~60.07%,核型类型均为2B,进化程度中等,处于由对称至不对称的过渡阶段,其核型进化的趋势大致为：‘Pink Passion’→‘Red River’→‘Gold River’→‘White River’→‘Castor’→‘上农红台阁’。聚类结果显示,遗传距离为15时可将所有品种分为两类,第一类包含‘Gold River’‘上农红台阁’和‘Castor’; 第二类包含‘Pink Passion’‘Red River’和‘White River’,其核型参数的差异可作为香雪兰品种分类和鉴定的辅助依据,为其细胞学研究和育种实践提供理论参考。     

Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Isolation and Characterization of a Cysteine Protease of Freesia Corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M _r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

六个不同类型荞麦花花粉粒形态的电镜观察比较研究

对二倍体甜荞长花柱类型 (ES2 s)、二倍体甜荞短花柱类型 (ES2 S)、四倍体甜荞长花柱类型 (ES4s)、四倍体甜荞短花柱类型 (ES4S)、四倍体有翅细野荞 (GR4HI)、四倍体无翅细野荞 (GR4HO)等 6个不同荞麦类型花的花粉形态学进行了电镜观察。结果表明 :这些荞麦类型的花粉粒都是椭圆形 ,都有网状纹饰、3孔沟等特征。二倍体甜荞染色体加倍后形成的四倍体甜荞 ,相对于二倍体甜荞而言 ,花粉粒显著增大和明显变圆。无论二倍体还是四倍体甜荞 ,其长花柱型花的花粉粒都比其对应短花柱型花的花粉粒要小。尽管四倍体甜荞和四倍体细野荞在染色体数目和倍性上一样 ,但是它们在花粉粒的大小上有显著差异。四倍体细野荞有翅类型和无翅类型的花粉粒大小和形态差异不显著     

The development of cysteine proteases in freesia corms during responses to chilling

Freesia protease (FP)-A has been found in regular freesia corms (Kaneda et al., Biosci. Biotechnol. Biochem. 61 (1997) 1554). New corms were generated from original corms that were kept for several months at 4°C. In this study, two proteases (FP-B and FP-C) have been found to new corms kept for 6 months at 4°C, and have increased during new corms enlargement.

 FPs were purified from the extracts of new corms, and the M_r of those were 24k (A), 25k (B), and 24.5k (C) by SDS-PAGE, respectively.

 The N-terminal sequences of FPs were identical to those of papain with respect to the conservative residues of cysteine protease. The sequence of FP-A was identical with those of FP-B within 20 residues of its N-terminal. It may be possible that FP-B was produced by some post-translational modifications from FP-A during the chilling. On the other hand, N-terminal sequence of FP-C was different from those of FP-A and FP-B. It was explained that FP-C was a new protease of freesia corm.     

Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

We have detected a flavonoid 3[prime],5[prime]-hydroxylase (F3[prime],5[prime]H) in the microsomal fraction of Petunia hybrida flowers. Activity varied with the development of flowers, peaking immediately prior to and during anthesis, but was absent in mature flowers. F3[prime],5[prime]H activity in flower extracts from genetically defined floral color mutants correlated strictly with the genotypes Hf1 and Hf2. No activity was detected in flowers from mutants homozygous recessive for both alleles. F3[prime],5[prime]H activity was dependent on NADPH and molecular oxygen; there was only slight activity with NADH. The enzyme catalyzes the hydroxylation of 5,7,4[prime]-trihydroxyflavonone at the 3[prime] and 5[prime] positions, and of 5,7,3[prime],4[prime]-tetrahydroxyflavonone and dihydroquercetin at the 5[prime] position. Hydroxylase activity was inhibited by plant growth regulators (1-aminobenzotriazole and tetcyclacis) and by CO, N-ethylmaleimide, diethyldithiocarbamate, and cytochrome (Cyt) c. Activity was not affected by diethylpyrocarbonate or phenylmethylsulfonyl fluoride, but was enhanced by 2-mercaptoethanol. A polyclonal antibody that inhibits higher plant NADPH-Cyt P450 reductase inhibited the F3[prime],5[prime]H. The data are consistent with the suggestion that the P. hybrida F3[prime],5[prime]H is a monooxygenase consisting of a Cyt P450 and a NADPH-Cyt P-450 reductase. Cyts P450 were detected in microsomal membranes and in solubilized detergent extracts of these membranes. F3[prime],5[prime]H activity was sensitive to low concentrations of all detergents tested, and therefore solubilization of the active enzyme was not achieved. Reaction products other than flavanones were observed in F3[prime],5[prime]H assays and these may be formed by enzymic oxidation of flavanones. The possibility of a microsomal flavone synthase of a type that has not been described in P. hybrida is discussed.     

Changes in abscisic acid levels during dormancy release in freesia corms

A high level of free-abscisic acid (ABA) was detected when corms were still in deep dormancy. The level of free-ABA decreased as the corm dormancy disappeared and increased temporarily after complete release from dormancy. A gradual slight increase of bound-ABA was observed during dormancy release.Treatment of dormant corms with benzyladenine (BA) increased sprouting but the sprouts did not show normal growth. Ethylene treatment induced complete sprouting and subsequent normal growth. Changes in ABA levels and ethylene production are discussed in relation to dormancy release in freesia corms.     

小苍兰种质遗传多样性的ISSR分析

利用ISSR(Inter Simple Sequence Repeat)分子标记对12份小苍兰(Freesia refracta)种质进行了遗传多样性分析研究。从34条ISSR引物中筛选出了12条适宜的引物。这12条引物中每条引物可扩增出5~11条DNA片段,共扩增了96个条带,其中多态性片段62条,平均每条引物可产生5.2条多态性片段,多态性条带比率(PPB)为64.6%。经NTSYS-pc分析,12份小苍兰种质间的遗传距离(GD)的变化范围为0.123~0.907,平均为0.442。根据Nei’s相似系数建立了UPGMA聚类图,在相似系数为0.56时,可将紫色花系的小苍兰种质与其它种质分开,形成两个组。结果表明,ISSR分子标记可有效地分析小苍兰种质资源的遗传多样性和亲缘关系,为小苍兰的杂交育种和新品种保护提供理论基础。     

The mode of inheritance in tetraploid cut roses

Tetraploid hybrid tea roses (Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F(1) progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al. (Theor Appl Genet 122:489-500, 2010) allowed assigning numbers to each of the linkage groups of both maps and including small linkage groups. So far, the possibility of using marker-assisted selection in breeding of tetraploid cut roses and of other species with a tetrasomic or partly tetrasomic inheritance, is still limited due to the difficulties in establishing marker-trait associations. We used these tetraploid linkage maps to determine associations between markers, two morphological traits and powdery mildew resistance. The knowledge on inheritance and marker-trait associations in tetraploid cut roses will be of direct use to cut rose breeding.     

四种简易切花保鲜剂比较试验

以瓶插寿命、鲜重及水分平衡值变化为指标,探讨由蔗糖、食盐、酒精、阿司匹林、食醋、青霉素等日常用品组成的四种配方的简易保鲜剂,对香石竹、非洲菊和玫瑰鲜切花的保鲜效果。结果表明,四种保鲜剂中,以配方B(1 L水+10 g蔗糖+10 g食盐+10 ml酒精)保鲜效果较为理想,能改善花枝的吸水状况,延长鲜切花水分平衡时间,从而延长鲜切花瓶插寿命。     

Expression of a polyubiquitin promoter isolated from Gladiolus

A polyubiquitin promoter (GUBQ1) including its 5′UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5′ and 3′ intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5′UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5′UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.     

矮牵牛(Petunia hybrida)开花过程中的可溶性蛋白质分析

在进行矮牵牛(Petunia hybrida)光周期诱导开花的过程中,对叶子中的可溶性蛋白质进行了分析,其中有4条与开花有关的特异蛋白,分子量分别为49.45 kD(a)、35.45 kD(b)、17.98 kD(c)和11.74 kD(d).在不开放的花苞中不含有蛋白质a和d,只有这4种蛋白质全出现时,才能形成花苞并且开放.花开了以后,蛋白质c和d就消失.即使在开花的植株中,各组织中的蛋白质也是不同的.茎中完全不含有蛋白质c和d,叶子和花中的蛋白质组成也是不同的.     

Purification of flavanone 3 beta-hydroxylase from Petunia hybrida: antibody preparation and characterization of a chemogenetically defined mutant

Flavanone 3 beta-hydroxylase from Petunia hybrida has been purified to apparent homogeneity utilizing an improved purification protocol including chromatography of the partially purified enzyme on hydroxyapatite, chromatofocusing, and hydrophobic interaction chromatography on phenyl-Superose. The specificity of mouse and rabbit polyclonal antisera directed to flavanone 3 beta-hydroxylase was demonstrated by Western blotting and immunotitration with crude extracts from wild-type flowers of P. hybrida and with the purified enzyme. Cross-reactivity was observed with flavanone 3 beta-hydroxylase from extracts of illuminated parsley cells. A Petunia mutant with white flowers, previously shown to lack 3 beta-hydroxylase activity and to accumulate flavanone glycosides, showed complete absence of the enzyme protein.     

Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy

Modern biotechnology has developed powerful tools for genetic engineering and flower colours are an excellent object to study possibilities and limitations of engineering strategies. Osteospermum hybrida became a popular ornamental plant within the last 20 years. Many cultivars display rose to lilac flower colours mainly based on delphinidin-derived anthocyanins. The predominant synthesis of delphinidin derivatives is referred to a strong endogenous flavonoid 3',5'-hydroxylase (F3'5'H) activity. Furthermore, since dihydroflavonol 4-reductase (DFR) of Osteospermum does not convert dihydrokaempferol (DHK) to leucopelargonidin, synthesis of pelargonidin-based anthocyanins is naturally not realised. In order to redirect anthocyanin biosynthesis in Osteospermum towards pelargonidin derivatives, we introduced cDNAs coding for DFRs which efficiently convert DHK to LPg. But neither the expression of Gerbera hybrida DFR nor of Fragaria x ananassa DFR - the latter is characterised by an unusual high substrate preference for DHK - altered anthocyanin composition in flowers of transgenic plants. However, chemical inhibition of F3'5'H activity in ray florets of dfr transgenic plants resulted in the accumulation of pelargonidin derivatives. Accordingly, retransformation of a transgenic plant expressing Gerbera DFR with a construct for RNAi-mediated suppression of F3'5'H activity resulted in double transgenic plants accumulating predominantly pelargonidin derivatives in flowers.     

Relationship between polyploidy and pollen self-incompatibility phenotype in Petunia hybrida Vilm

Self-incompatibility in Solanaceae is controlled by a single multiallelic locus, the S-locus. The S-allele associated ribonucleases (S-RNases) in the pistil are involved in pollen rejection. In this work, we analyzed two newly isolated lines of Petunia hybrida, termed PB and PF. They both had the same set of S-RNases (SB1- and SB2-RNases), however the PB was a self-incompatible diploid while PF was a self-compatible tetraploid. Cross pollination tests between PB and PF indicated diploid pollen from PF lost the incompatibility phenotype. In order to clarify the effects of polyploidy on pollen phenotypic change, we artificially induced tetraploid plants from a diploid SB1SB2 heterozygote (= PB) and a diploid SB1SB1 homozygote. The obtained SB1 SB1SB1SB1 homoallelic tetraploid remained self-incompatible, whereas the SB1SB1SB2SB2 heteroallelic tetraploid became self-compatible. These data suggested that the diploid heteroallelic pollen lost the incompatibility phenotype and had the characteristics of self-compatibility with SB1SB2 style.     

A protocol for fertility restoration of F<Subscript>1</Subscript> hybrid derived from <Emphasis Type="Italic">Lilium</Emphasis> × <Emphasis Type="Italic">formolongi</Emphasis> ‘Raizan 3’ × Oriental hybrid ‘Sorbonne’

The present study was an attempt to develop an in vitro colchicine chromosome doubling protocol to restore the fertility of an F₁ interspecific hybrid in Lilium. Basal scale segments of Lilium?×?formolongi?×?Oriental hybrid (FO) bulblets were pre-cultured for three durations (6, 15 and 25 days) and soaked in three colchicine concentrations (0.00, 1.25 and 2.50 mM) for 18, 24 and 36 h. To separate mixoploids, three cycles of adventitious bud induction were performed. The ploidy levels of the surviving plantlets were detected by flow cytometry at 30–31 weeks after induction and were confirmed by chromosome counts. The results indicated that the pre-culture duration, colchicine concentration and exposure time all had significant impacts on the tetraploid induction rate. The preferred procedure was to pre-culture the segments for 6 days and then treat them with 1.25 mM colchicine for 24 h. The morphological traits of rosette leaves were significantly different between the tetraploid and diploid plants. The adult tetraploid plants had considerably longer and wider leaves, larger flowers, and delayed flowering time (8 days later) than did diploid plants. Pollen viability tests and backcross trials of FO hybrids demonstrated fertility restoration at the tetraploid level. This protocol provides a feasible method for inducing fertile tetraploid FO hybrids for further breeding.     

连翘花蕾与花的挥发性成分研究

本文采用顶空固相微萃取和气相色谱质谱联用(HS-SPME-GC-MS)技术比较分析了连翘花蕾和花的挥发性成分。从连翘花蕾和花中共鉴定了83个化合物,其中40个化合物是共有的。连翘花蕾的挥发性成分以烃类(41.04%)为主,而连翘花的挥发性成分以醇、醛和酮类化合物(26.46%)为主;酯类化合物在花蕾中的含量为8.11%,在花中下降到2.12%;而酸类化合物则从3.56%上升到5.18%。结果表明,随着花蕾的开放,酯类成分逐渐被水解为酸。     

矮牵牛花期一些生理指标的变化

选择了 3种颜色的矮牵牛 (PetuniahybridaVilm) :粉红色、杂色和红色 ,将其开花过程分为 4个时期 :未出现花芽、花芽期、花蕾期和开花期 ,测定各时期MDA、可溶性糖、激素水平和多胺含量等指标的变化。结果表明 ,从无花芽期到开花期MDA含量有所升高 ;可溶性糖含量呈现降低的趋势。在粉红色的矮牵牛叶片中 ,IAA含量在开花期升高 ;GA含量在无花芽期和花芽期时较高 ;而ZRs则在花蕾期较低 ,在开花期时含量上升。 3种多胺含量的变化不同 ,腐胺在整个花期略有上升 ,精胺和亚精胺则略有下降