CONSTRUCTION OF A GENETIC LINKAGE MAP FOR PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) BASED ON SEQUENCE‐RELATED AMPLIFIED POLYMORPHISM AND SIMPLE SEQUENCE REPEAT MARKERS1

Molecular markers and molecular genetic maps are prerequisites for molecular breeding in any plant species. A comprehensive genetic linkage map for cultivated Porphyra haitanensis T. J. Chang et B. F. Zheng has not yet been developed. In this study, 157 double haploid (DH) lines [derived from a YSIII (wildtype) × RTPM (red‐type artificial pigmentation mutant) cross] were used as a mapping population in P. haitanensis. A total of 60 pairs of sequence‐related amplified polymorphism (SRAP) primers and 39 pairs of simple sequence repeat (SSR) primers were used to detect polymorphisms between the two parents. Fifteen SRAP and 16 SSR polymorphic primer pairs were selected to analyze the DH population. A linkage genetic map comprising 67 SRAP markers and 20 SSR markers in five linkage groups, with a total length of 830.6 cM and an average of 10.13 cM between markers, was constructed. The markers were distributed evenly in all linkage groups without clustering. The linkage groups comprised 12–23 markers ranging in length from 134.2 to 197.3 cM. The estimated genome length of P. haitanensis was 942.4 cM, with 88.1% coverage. This is the first report of a comprehensive genetic map in P. haitanensis. The map presented here will provide a basis for the development of high‐density genetic linkage maps and lay the foundation for molecular breeding work in P. haitanensis.     

利用脱落酸和冷适应提高坛紫菜丝状体种质的冻存效率

为验证脱落酸(ABA)和冷适应能否提高紫菜丝状体种质的冷冻保存效率,研究以坛紫菜(Pyropia haitanensis)自由丝状体为材料,在20℃,光强40μmol·photons/(m2·s),在光周期12L﹕12D条件下以ABA(100μmol/L)处理48h或4℃暗培养6d,随后进行–80℃冷冻保存实验.通过连...     

应用超滤技术提取坛紫菜多糖的研究

以截留分子量为50 KD的超滤膜对坛紫菜粗多糖提取液进行超滤分离。结果表明,超滤对坛紫菜粗多糖提取液具有较好的脱色效果,脱色率达91.65%。超滤技术能很好地保留坛紫菜粗多糖中的生理活性物质,浓缩了坛紫菜粗多糖提取物,使粗多糖中总糖含量从超滤前的34.95%提高到52.07%,明显提高了坛紫菜粗多糖的糖含量,同时进一步纯化了样品。     

坛紫菜别藻蓝蛋白α亚基基因的原核表达与鉴定

通过PCR的方法从重组质粒pMD-apcAB中扩增坛紫菜别藻蓝蛋白α亚基基因(apcA),并将其克隆到高效表达外源基因的原核表达质粒pTO-T7。将构建好的质粒导入表达型大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行Western-blot和质谱鉴定。结果显示:apcA全长486bp,表达的α亚基(apcA)为带有原核表达载体T7g10的12个起始氨基酸的融合蛋白,其分子量约为19.7KD。0.5mmol/L的IPTG在37℃诱导6h时,apcA的表达量达到最大,达菌体总蛋白50%以上。Western-blot和质谱鉴定的结果表明获得的融合蛋白为重组坛紫菜别藻蓝蛋白α亚基。       

New insights into the biodiversity and generic relationships of foliose Bangiales (Rhodophyta) in Iceland and the Faroe Islands

Foliose species of the Bangiales (Porphyra sensu lato) have a long history of study in the N Atlantic, but there are still regions, especially in the northern parts of the N Atlantic that need more attention. A molecular study using rbcL and cox1 sequences was undertaken to assess the diversity of foliose Bangiales species in Iceland and the Faroe Islands. Herbarium collections from the intertidal and subtidal of Iceland (summer and winter) and the Faroe Islands (all seasons) revealed a total of 13 species (11 common to both areas), which were referred to four of the genera recognized in a recent two-gene global phylogeny. Boreophyllum birdiae, Porphyra dioica, P. linearis, P. purpurea, P. umbilicalis, Pyropia ‘leucosticta’ A, Pyropia njordii Mols-Mortensen, J. Brodie & Neefus, sp. nov., Wildemania amplissima and W. miniata were common to both areas, while Pyropia thulaea and Wildemania abyssicola (Kjellman) A. Mols-Mortensen & J. Brodie, comb. nov. (=Porphyra abyssicola Kjellman) were reported from Iceland but not from the Faroe Islands; Porphyra sp. FO and Pyropia elongata were reported from the Faroe Islands but not from Iceland. Boreophyllum birdiae is reported for the first time for Iceland and Porphyra sp. FO is reported for the first time for the Faroe Islands. Pyropia njordii is described from the Faroe Islands and is also recorded for Iceland, Greenland, New England, USA and Nova Scotia, Canada. A total of 25 foliose Bangiales species are now reported from the N Atlantic and these results demonstrate the importance of investigating as many areas as possible to reach a more complete understanding of species diversity and distribution.     

坛紫菜藻红蛋白的规模化制备

为了优化坛紫菜粗提工艺以减轻纯化的压力,研究了我国主要经济红藻之一坛紫菜藻红蛋白大规模制备方法。实验采用“溶胀+组织捣碎”法破碎坛紫菜叶状体细胞,对比了多次硫酸铵梯度盐析对破碎液中藻红蛋白的影响,并进一步用羟基磷灰石层析制备藻红蛋白,最后对所得蛋白做了光谱和电泳鉴定。结果表明,经过4次盐析,藻红蛋白吸收光谱纯度达到0.9 (A564/A280),每次的最佳盐析浓度分别为15％、50％、10％和40％;7 kg阴干紫菜经过4次盐析和1次羟基磷灰石层析后可获得507.82 mg藻红蛋白 (A564/A280＞     

坛紫菜菌解液对植物生长和抗性指标的影响

利用琼胶降解菌处理坛紫菜粉末,降解其细胞壁多糖,释放内容物,并获得菌解液,研究不同稀释度坛紫菜菌解液对受体植物蚕豆出芽和生长,以及大豆和番茄幼苗抗性相关指标的影响.结果显示:(1)随琼胶降解菌处理时间的延长,菌解液中还原糖含量逐渐增高.(2)3.33%的菌解液对蚕豆种子萌芽促进效果最好,而2%的菌解液能增加蚕豆叶绿素含量,促进其幼苗生长.(3)3.33%的菌解液能有效提高大豆离体子叶的植保素含量,迅速增加番茄叶片表皮条的H2O2的释放量,提高苯丙氨酸解氨酶活性.研究表明,琼胶降解菌能使坛紫菜细胞壁中的营养物质释放,并产生某些激发物质,从而使坛紫菜菌解液能够促进受体植物的种子发芽和幼苗生长,并能有效诱导植物的抗性.     

坛紫菜（Porphyra Haitanensis）中抑菌活性肽的制备与初步纯化

目的：提取坛紫菜中水溶性蛋白,并对其进行初步纯化和抑菌活性影响因素研究。方法：坛紫菜水溶性蛋白胃蛋白酶在37℃、pH1.8条件下酶解3h,再经超滤、Bio-Gel P-10和DEAE Sephadex A-50层析纯化步骤得到一定分子量范围的多肽混合物。采用平板打孔法和对金黄色葡萄球菌的抑制作用,跟踪测定活性多肽纯化过程及其活性影响因素。结果：SDS-PAGE测定结果表明该抗菌多肽分子量介于43.0KD~66.2KD之间。它对金黄色葡萄球菌生长的抑制作用随着温度的升高逐渐减弱,5%EDTA、5%柠檬酸、5%维生素C及25%二甲基亚砜对它的抑菌活性有协同作用,而5%维生素E则对它的抑菌活性有拮抗作用。结论：从坛子菜水溶性蛋白中初步纯化得到的分子量介于43.0KD~66.2KD的多肽,对金黄色葡萄球菌的生长有明显地抑制作用,并且它的抑菌活性受到温度,部分有机酸和维生素的影响。     

坛紫菜叶状体的无菌化培养及其应用

对坛紫菜叶状体的无菌处理方法进行了优化,并利用紫菜外生菌对无菌处理后的紫菜叶片进行了人工感染。经0.7%KI和0.1%(w/v)氨苄青霉素对紫菜叶片进行预处理后,利用氨苄青霉素、硫酸链霉素、新霉素、庆大霉素及卡那霉素等5种抗生素及其不同组合对紫菜叶片进行处理,筛选出最佳抗生素组合、浓度和培养条件。结果表明:氨苄青霉素(终浓度300μg/mL)、卡那霉素(终浓度100μg/mL)与庆大霉素(终浓度100μg/mL)3种抗生素组合对坛紫菜叶状体进行无菌处理18 h,对紫菜细胞的毒害较小,并且3种抗生素合用对89.5%紫菜外生细菌的抑菌率达80%以上;外生菌感染结果研究表明:用105/mL真菌孢子液和细菌菌液回染紫菜,培养22 d后,实验组紫菜生长状况较对照组好,对照组紫菜出现褪色。用108/mL真菌孢子液分别回染健康紫菜、穿刺紫菜(用灭菌刀片在紫菜表面划1～2 mm的伤口),分别在18℃、28℃和35℃条件下培养。实验结果表明28℃和35℃高温和穿刺均会影响无菌紫菜健康生长,且加菌紫菜在高温和穿刺条件下,则会出现明显病斑,而18℃培养的加菌紫菜则生长良好。