A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell-boring, filamentous sporophytes and free-living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase-specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase-specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase-specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase-specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte-specific cDNAs and two by the gametophyte-specific cDNAs were identified by their similarity to databank entries.     

Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species. This revised version was published online in June 2006 with corrections to the Cover Date.     

大孔吸附树脂对紫锥菊提取物中菊苣酸分离纯化的研究

采用大孔吸附树脂分离纯化紫锥菊提取物中免疫活性成分菊苣酸,7％(v／v)的甲醇-水溶液洗脱,HPLC法对产品中菊苣酸含量进行检测分析。该法能把紫锥菊提取物中菊苣酸含量(4％)提高9倍左右,回收率达到95％以上,且操作简单,可用于菊苣酸的分离纯化。     

紫果猕猴桃嫩枝多酚氧化酶活性与总酚含量变化

采用紫果猕猴桃嫩枝和叶柄为试材,分析全年中植株多酚氧化酶（PPO）活性和总酚含量的变化规律。结果表明,紫果猕猴桃植株PPO活性的最适pH为7.0;最佳底物为邻苯二酚,且浓度为0.16 mol/L;最佳温度为25 ℃。紫果猕猴桃植株生长期的PPO活性明显高于休眠期,植株总酚含量在4月、5月、9 月达最低。     

紫果猕猴桃幼胚愈伤组织诱导及植株再生

以紫果猕猴桃(Actinidia arguta var．purpurea)幼胚为外植体,诱导愈伤组织并进行植株再生。结果表明：不同的培养基和不同的培养条件对幼胚愈伤组织的诱导率及分化率不同;0．2mg／L ZT与0．5mg／L GA。配合使用有利于促进愈伤组织的诱导;7％蔗糖、600mg／L CH与400mg／L Gln都有利于促进愈伤组织的形成;在添加0．5mg／L 6-BA、0．05mg／L NAA与0．5mg／L GA3的MS培养基中植株的再生率达93．3％。     

象牙参种子的解剖学和组织化学研究

象牙参种子解剖学和组织化学的研究结果表明, 种子包括假种皮、种皮、外胚乳、内胚乳和胚。假种皮没有完全包被种子, 由约4~5 层薄壁细胞构成。种皮可以分为外种皮、中种皮和内种皮。外种皮由1 层表皮细胞构成, 细胞壁明显增厚;中种皮包括下皮层、半透明细胞层和3~4层细胞的色素层, 下皮层和色素层细胞均充满红棕色色素;内种皮由1 层体积小、壁局部增厚的砖形薄壁细胞构成。种子在珠孔端分化出珠孔领、孔盖和种阜状结构, 珠孔领为同形型, 孔盖不具石细胞硬层。合点区内种皮出现缺口, 缺口间充满合点区色素细胞, 其整体轮廓成新月形。外胚乳可分为厚区与薄区两部分, 外胚乳细胞壁平直, 细胞内充满淀粉。内胚乳细胞主要含蛋白质, 也有少量脂类物质, 细胞界限不清楚。胚棒状, 两端略膨大, 含大量脂类物质, 也含蛋白质和多糖。     

野牛草幼穗愈伤组织的诱导及植株再生

以野牛草[Buchloe dactyloides(Nutt．)Engelm．]幼穗为外植体,建立了愈伤组织诱导、继代培养和植株再生体系。结果表明,雌穗比雄穗难以脱分化形成愈伤组织;小于8mm雄幼穗在2mg／L2,4-D培养基上的愈伤组织诱导率为80．0％～86．8％;添加10mg／L AgNO3对愈伤组织诱导率影响不明显,但可改善愈伤组织质量。2mg／L 2,4-D结合0．1mg／L 6-BA的培养基有利于愈伤组织的继代培养;继代超过3次、继代间隔超过3周,愈伤组织分化能力明显下降。雄穗愈伤组织在含1．0mg／L 6-BA培养基上,弱光条件下分化出芽的频率较高,达31．8％～35．0％;附加3％麦芽糖既可减轻褐化程度,又利于丛生芽的分化。分化苗在1/2MS 0．3mg／L IBA培养基上的生根率为62．5％。     

Analysis of a chalcone synthase mutant in Ipomoea purpurea reveals a novel function for flavonoids: amelioration of heat stress

Flavonoids are thought to function in the plant stress response and male fertility in some, but not all, species. We examined the effects of a self-fertile chalcone synthase null allele, a, for the effects of heat and light stress on fertilization success and flower production in Ipomoea purpurea. Pollen recipients and pollen donors of both homozygous genotypes exhibit reduced fertilization success at high temperatures, indicating that high temperature acts as a stress-lowering fertilization success. Homozygous aa individuals exhibit reduced male and female fertilization success, compared to AA individuals, at high temperatures but not at low temperatures. In addition, aa individuals produce fewer flowers than AA individuals at low temperatures, but not at high temperatures. These results suggest that flavonoids alleviate heat stress on fertilization success. They also suggest that pleiotropic effects at the A locus may explain the low frequency of the a allele in natural populations.     

洮河上游紫果云杉种群结构特征

该研究以洮河上游尕海-则岔自然保护区、卡车林区和冶力关林区的紫果云杉(Picea purpurea)天然种群为研究对象, 通过样地调查和数据统计, 绘制种群结构图, 编制静态生命表, 拟合并分析存活曲线, 运用数量化方法研究种群动态, 揭示种群生存现状, 预测种群发展趋势, 以期为该物种的保护、管理及结构恢复提供理论依据。结果显示: 3个林区紫果云杉种群自然更新能力强, 幼苗、幼树储量丰富, 幼小龄期死亡率普遍偏高; 尕海-则岔种群存活曲线符合Deevey-III型, 种群稳定结构完整, 卡车林区和冶力关林区种群存活曲线均符合Deevey-II型, 且均出现了局部衰退; 3个林区种群动态指数均大于0, 说明种群均属于增长型, 增长潜力为尕海-则岔>卡车>冶力关; 受随机干扰时卡车林区紫果云杉最敏感, 冶力关次之, 尕海-则岔种群最稳定。该研究表明: 竞争和自疏作用是造成紫果云杉幼小龄级个体存活率偏低的普遍因素, 3个林区不同的生存状况反映了紫果云杉种群在不同生境及生活史下生存能力的差异, 保护幼苗生存环境并提高幼苗质量和存活率是种群更新和发展的关键。尕海-则岔紫果云杉生存良好, 种群生存状况主要受自身生物学特性和环境因子的影响; 卡车林区主要受人为影响, 种群结构遭到破坏; 冶力关林区受分布限制, 造成种群结构不稳定, 须采取一定的人工措施来促进种群更新与增长。