苏铁的化学成分研究

对苏铁(Cycas revoluta)的化学成分进行研究,采用多种色谱技术(硅胶、氧化铝和Sephadex LH-20等)从苏铁茎的乙酸乙酯部位分离得到16个化合物,其结构由HR-ESI-MS、1H和13CNMR等波谱学方法鉴定为5,6-去氢柳杉酚(1)、cunningine A (2)、6-羟基-5,6-去氢柳杉酚(3)、6α-羟基-7-氧代弥罗松酚(4)、ligballinol (5)、xanthoxyol (6)、callislignan A (7)、(2R,3R)-bis[(4-hydroxy-3-methoxyphenyl) methyl]-1,4-diacetate (8)、开环异落叶松脂醇(9)、二氢山柰酚(10)、4'-甲基木犀草素(11)、5-methoxypinosylvin (12)、N-benzoylphenyl alaninol(13)、(E,4R)-4-hydroxy-4,5,5-trimethyl-3-(3-oxobut-1-enyl) cyclohex-2-enone (14)、3-hydroxy-5α,6α-epoxy-β-io-none (15)和acuminantin (16)。以上化合物均为首次从苏铁属植物中分离得到,其中化合物13和16为首次以天然产物报道。本研究还首次发现了苏铁属植物中含有松香烷型二萜类(1～4)和芪类(12)化合物。化合物11具有中等的体外α-葡萄糖苷酶抑制活性。     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

苏铁白化苗叶片形态与结构观察

本文对苏铁(Cycas revoluta)白化苗与绿色正常苗的形态特征及叶片显微结构进行比较观察。结果表明,两者之间存在许多显著且稳定的差异。     

Estragole (4-allylanisole) is the primary compound in volatiles emitted from the male and female cones of Cycas revoluta

The genus Cycas (Cycadaceae; gymnosperm) have circumstantially been considered to be wind-pollinated. The cones of Cycas revoluta Thunb., however, emit a strong unpleasant odor. The chemical profiles of floral scents often correlate with various pollination modes (pollinators). We collected and analyzed the volatiles emitted from male and female cones of C. revoluta native to Iriomote and Yonaguni Islands, Japan. The analyses indicated that estragole (4-allylanisole) dominated in the volatiles (67.0–92.7%), with small amounts of other benzenoids, e.g., anethole, methyl salicylate, methyl eugenol, and ethyl benzoate. Several fatty acid esters were also detected in the samples from Iriomote Island. The function of estragole in the reproductive biology of C. revoluta is discussed.     

苏铁的引种栽培与生长状况调查

厦门园林植物园自20世纪60年代开始开展苏铁的迁地保育工作,对园内引种苏铁的栽培现状、生物学特性以及生长状况的调查表明,厦门园林植物园引种种植的苏铁长势良好,花期为5~6月,种子10~11月成熟;植株生长缓慢,干高年均生长量3.026cm,地径年均生长量0.726cm。     

In vitro organogenesis from diploid tissues of Cycas revoluta Thunb.

Leaf base and mesocotyl explants derived from in vitro-grown seedlings of Echinochloa colona were cultured on Murashige and Skoog's (MS) medium containing various concentrations of benzyladenine (BA), -naphthaleneacetic acid (NAA) and kinetin. Leaf base and mesocotyl segments exhibited optimal morphogenetic response by using 6.66 M BA with 2.68 M NAA. Induction of rooting from regenerated shoots was readily achieved in half strength MS medium without organics and growth regulators. Histological studies revealed the sequence of shoot bud regeneration in the monocot system. The in vitro-raised plants were established in chromite minewaste.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - MS medium Murashige & Skoog's (1962) medium     

苏铁(Cycas revoluta Thunb.)种子的胚与胚乳解剖结构研究

对苏铁(Cycas revoluta Thunb.)种子的胚和胚乳组织进行了解剖研究。结果表明:苏铁种子为有胚乳种子,兼有胚乳和外胚乳,成熟时具直立型胚。胚乳的表层细胞含有角蜡质,胞核大,不含淀粉粒;中层细胞胞核明显;内层细胞胞核不明显,富含淀粉颗粒,淀粉粒单脐点明显。胚孔端的胚乳内陷成一凹槽,似贮藏窖。成熟的子叶胚为倒生胚胎,位于胚乳细胞解体后形成的囊腔中,子叶胚长度在胚乳中占到种子的1/3至2/3,已达到生理成熟阶段。双子叶直立,半合生。胚状体基部呈喙状突起,喙状突起下端连着一根肠叠着的丝状吸器,吸器基部连着一个小气囊。胚芽由顶端分生组织和数枚真叶组成,此时真叶已具羽状叶原基和绒毛原始体。在胚状体中发现有长管细胞及螺纹加厚的导管,在子叶中脉有数条并列的螺环纹导管。     

苏铁白化苗叶片细胞的超微结构

本文系苏铁白化苗研究系列报道之二。应用超薄切片和电镜观察技术对苏铁白化苗叶片细胞的超微结构观察,结果表明,苏铁白化苗叶片细胞的超微结构与绿苗的相比有明显不同,前者的细胞质含量稀少,未见有结构完整的叶绿体和线粒体,但见有呈支解、游离状的类似叶绿体片层的膜结构和内嵴几乎完全消失的退化线粒体存在;后者的细胞结构正常。     

苏铁Cycas revoluta珊瑚根内生真菌的多样性

苏铁类植物是目前发现的唯一一类具有珊瑚根结构的裸子植物,是探究植物-微生物互作的理想材料。本研究通过高通量测序技术对中国13个地区栽培苏铁Cycas revoluta珊瑚根中的内生真菌组成及多样性进行分析,结果表明苏铁内生真菌多样性较高,但各地区间内生真菌的组成及多样性差异不显著,显示苏铁和珊瑚根内生真菌的共生与栽培地无关。     

Factors affecting shoot regeneration from zygotic embryo and seedling explants of Cycas revoluta thunb

Summary Adventitious shoot induction from zygotic embryo and seedling explants of Cycas revoluta was studied, with reference to both nitrogen formulation of the medium and light. The presence of nitrate as a sole source of nitrogen in Klimaszewska and Keller (KK) medium did not promote shoot induction; on the other hand, shoot induction was promoted by the use of ammonium-containing media, i.e., Schenk and Hildebrandt (SH) and Murashige and Skoog (MS). In the case of zygotic embryos, the percentage of responding explants and the number of regenerated shoots were significantly higher on SH medium than on MS medium. With seedling explants, shoot induction was not affected by the use of SH medium versus MS medium. Photoperiod also influenced bud formation and development. The absence of light during shoot induction stimulated callus formation and very few differentiated shoots. Shoot induction from zygotic embryos was positively affected by the application of 4- and 8-wk photoperiod exposures, the combination SH medium/8-wk photoperiod exposure giving the best results in terms of shoot induction and development. Rooting of shoots was improved by their culture in medium containing NAA.     

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae)

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400?bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.     

Pollen-tube guidance: beacons from the female gametophyte

The sperm cell of a flowering plant cannot migrate unaided and it must be transported by the pollen-tube cell before successful fertilization can occur. The pollen tube is precisely guided to the target female gametophyte, the embryo sac, which contains the egg cell. The mechanism that precisely directs the pollen tube through the pistil to the female gametophyte has been studied for more than a century. There has been controversy over whether a diffusible signal attracts the pollen tube or whether female tissues define its path. Emerging genetic and physiological data show that the female gametophyte produces at least two directional signals, and that at least one of these signals is diffusible and derived from the two synergid cells.     

beta-D-Glucosidase from seeds of Japanese cycad, Cycas revoluta Thunb.: properties and substrate specificity

beta-D-Glucosidase was purified from seeds of Japanese cycad by dialysis, chromatography on CM-Sepharose CL-6B, gel filtration on Biogel P-200, and chromatofocusing. By chromatofocusing, beta-D-glucosidase was separated into four components whose isoelectric points were in a very narrow range (7.43-7.68). All these components were glycoproteins. The main component (pI = 7.59) was homogeneous on gel isoelectric focusing, and was crystallized from ammonium sulfate solution. The molecular weight of the crystalline preparation was determined to be 137,000 by gel filtration, and 67,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating the main component was composed of two subunits with the same molecular weight. The amino acid composition and sugar content of the main component were also determined. All four components hydrolyzed not only o-nitrophenyl beta-D-glucopyranoside but also o-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-fucopyranoside, and o-nitrophenyl beta-D-xylopyranoside. Hydrolysis rates of each substrate by the four components were quite similar. Mixed substrate experiments using crystalline preparation proved that a single active site was responsible for the hydrolysis of these substrates.     

Identification and characterization of a bactericidal and proapoptotic peptide from Cycas revoluta seeds with DNA binding properties

Nowadays, novel pharmacies have been screened from plants. Among them are the peptides, which show multiple biotechnological activities. In this report, a small peptide (Ala–Trp–Lys–Leu–Phe–Asp–Asp–Gly–Val) with a molecular mass of 1,050 Da was purified from Cycas revoluta seeds by using reversed‐phase liquid chromatography. This peptide shows clear deleterious effects against human epidermoid cancer (Hep2) and colon carcinoma cells (HCT15). It caused inhibition of cancer cell proliferation and further disruption of nucleosome structures, inducing apoptosis by direct DNA binding. A remarkable antibacterial activity was also observed in this same peptide. Nevertheless, no significant lysis of normal RBC cells was observed in the presence of peptide. Additionally, an acetylation at the N‐termini portion is able to reduce both activities. Bioinformatics tools were also utilized for construction of a three‐dimensional model showing a single amphipathic helix. Since in vitro binding studies show that the target of this peptide seems to be DNA, theoretical docking studies were also performed to better understand the interaction between peptide and nucleic acids and also to shed some light on the acetyl group role. Firstly, binding studies showed that affinity contacts basically occur due to electrostatic attraction. The complex peptide‐ssDNA was clearly oriented by residues Ala¹, Lys³, and Asp⁶, which form several hydrogen bonds that are able to stabilize the complex. When acetyl was added, hydrogen bonds are broken, reducing the peptide affinity. In summary, it seems that information here provided could be used to design a novel derivative of this peptide which a clear therapeutic potential. J. Cell. Biochem. 113: 184–193, 2012. © 2011 Wiley Periodicals, Inc.     

Occurrence and Localization of Phycoerythrin in Symbiotic Nostoc of Cycas revoluta and in the Free-Living Isolated Nostoc 7422

The phycobiliprotein phycoerythrin was localized in symbiotic and free-living Nostoc of the cycad Cycas using immunocytochemistry. In symbiotic Nostoc, phycoerythrin was associated with the thylakoid membranes of vegetative cells and absent from heterocysts. Similar cellular/subcellular localization was observed between symbiotic Nostoc and the free-living Cycas isolate Nostoc 7422.     

Cloning and expression of a mannose-binding jacalin-related lectin from leaves of Japanese cycad (Cycas revoluta Thunb.)

Cycad leaf lectin (CRLL), a mannose-recognizing jacalin-related lectin (mJRL), was first cloned as a gymnosperm lectin and expressed. The cDNA sequence of CRLL (DDBJ, accession no. AB198328), coding 291 amino acid residues, has a tandem repeat of about 150 amino acids divided into N- and C-terminal domains as Japanese chestnut mJRL. Sequence alignment showed deletion and insertion of the sequence, and its putative carbohydrate-binding sites showed some differences from other JRLs. PCR analysis showed that this lectin was expressed in the cycad leaf but not in the root or seed. Recombinant CRLL (rCRLL) was expressed in Escherichia coli and purified by affinity chromatography after refolding procedures. Properties of active rCRLL appeared to be almost the same as those of native CRLL.     

Cloning and Expression of a Mannose-Binding Jacalin-Related Lectin from Leaves of Japanese Cycad (Cycas revoluta Thunb.)

Cycad leaf lectin (CRLL), a mannose-recognizing jacalin-related lectin (mJRL), was first cloned as a gymnosperm lectin and expressed. The cDNA sequence of CRLL (DDBJ, accession no. AB198328), coding 291 amino acid residues, has a tandem repeat of about 150 amino acids divided into N- and C-terminal domains as Japanese chestnut mJRL. Sequence alignment showed deletion and insertion of the sequence, and its putative carbohydrate-binding sites showed some differences from other JRLs. PCR analysis showed that this lectin was expressed in the cycad leaf but not in the root or seed. Recombinant CRLL (rCRLL) was expressed in Escherichia coli and purified by affinity chromatography after refolding procedures. Properties of active rCRLL appeared to be almost the same as those of native CRLL.     

New perspectives on the angiosperm female gametophyte

This review builds upon previous classifications of angiosperm female gametophytes but offers two new perspectives. Firstly, the course of development is compared to an algorithm: a predetermined set of rules that produces a mature female gametophyte. This analogy allows hypotheses to be developed as to what changes in the “developmental program” are responsible for variant forms of development. Secondly, the review recognizes that the four meiotic products of a megaspore mother cell have different genetic constitutions and may have conflicting interests. In most cases, only one member of a megaspore tetrad gives rise to a functional egg. This megaspore is called the germinal spore. The other members of the tetrad are called somatic spores. Somatic spores do not give rise to functional eggs and, therefore, cannot leave direct genetic descendants. Non-monosporic embryo sacs are genetic chimeras containing derivatives of more than one megaspore nucleus. Conflict may arise within such embryo sacs between the derivatives of the germinal megaspore nucleus and the derivatives of somatic megaspore nuclei. “Antipodal eggs” and chalazal “strike” are interpreted as evidence of this conflict. The behavior of somatic spores and their derivatives is often variable for different embryo sacs produced by the same sporophyte. This has created difficulties for existing classifications of embryo sac “types” because more than one type is sometimes recognized within a species. A new classification of developmental algorithms is presented that emphasizes the fate of the germinal spore and its derivatives.