石蒜属植物多糖的分离纯化

对五种石蒜属植物,安徽石蒜、中国石蒜、忽地笑、换锦花、石蒜多糖的提取、分离、纯化及其理化性质进行了研究。为石蒜属植物多糖开发利用提供依据,更为相关石蒜属植物多糖的最佳提纯过程和探讨该属种间亲缘关系提供资料。     

石蒜属植物分支系统学分析

基于37个形态学、解剖学、孢粉学和细胞学性状及解剖学性状之外的28个形态学、孢粉学和细胞学性状,分别对石蒜属进行分支系统学分析,试图建立石蒜属种间的系统发育关系。利用PAUP^*软件分别构建了最大简约树(MP),所得树的拓扑结构是一致的。同时,基于解剖学9个性状,对石蒜、换锦花、忽地笑、江苏石蒜、长筒石蒜、乳白石蒜、夏水仙、红兰石蒜、安徽石蒜、短蕊石蒜、中国石蒜11个种进行系统发育树构建,其结果也是支持上述系统发育树的。系统发育树结构结果表明,石蒜属16种明显聚为两大类:石蒜、玫瑰石蒜、稻草石蒜和江苏石蒜;广西石蒜、红兰石蒜、换锦花、香石蒜、夏水仙、长筒石蒜、安徽石蒜、中国石蒜、忽地笑、乳白石蒜、短蕊石蒜和陕西石蒜。除换锦花、红兰石蒜及江苏石蒜系统发育位置不同之外,大类群的划分与RAPD指纹图谱基本一致。类群一均属于石蒜亚属(Lycoris亚属)。类群二又可以聚为两小类:广西石蒜、红兰石蒜、换锦花、香石蒜、夏水仙归为一类;长筒石蒜、安徽石蒜、中国石蒜、忽地笑、乳白石蒜、短蕊石蒜和陕西石蒜归为一类。前一子类群除广西石蒜外,都属于整齐花亚属(Symman thus亚属)。后一子类群除长筒石蒜与安徽石蒜外,均属于石蒜亚属(Lycoris亚属)。因此,花冠整齐与否是一个重要的分类特征,但作为石蒜属植物亚属的划分依据,没有得到本研究支持。而在本文中,雄蕊与花被片的位置关系可以作为大分类群划分依据,能否依此来对石蒜属植物亚属进行划分,仍需探讨。另外研究表明叶微形态特征在研究种间亲缘关系时,具有一定的作用。而在种间亲缘关系鉴定时,出叶期不应成为重要的依据。同时研究还表明中国石蒜与忽地笑具有非常近的亲缘关系,与形态学研究一致。     

应用HPLC图谱进行石蒜属种间关系与分类研究

利用高效液相色谱(HPLC)对石蒜属及其近缘植物中国水仙共17份样品的生物碱进行检测,以各样品生物碱色谱峰的相对保留时间为变量,采用SPSS12.0中的Q型聚类法,得到树形图。聚类分析结果显示,17份样品聚成3类:第Ⅰ类包括长筒石蒜、安徽石蒜、鹿葱、换锦花、中国石蒜、香石蒜、乳白石蒜、红蓝石蒜8个种;第Ⅱ类由石蒜、稻草石蒜、江苏石蒜、忽地笑、玫瑰石蒜、矮小石蒜组成;中国水仙单独成第Ⅲ类。聚类结果与传统分类结果有较好的一致性,并支持鹿葱、玫瑰石蒜、安徽石蒜杂交起源的观点。外形相似的忽地笑与中国石蒜,矮小石蒜与石蒜HPLC图谱明显有别,表现出较远化学亲缘关系。表明HPLC化学图谱可用于石蒜属及其近缘植物中国水仙的分类和鉴别研究,并为石蒜属植物指纹图谱研究和质量控制提供重要参考。     

中国石蒜属种间亲缘关系ITS序列分析

本文利用核糖体DNA内转录间隔区(ITS)序列对石蒜属13个种(含变种)的亲缘关系进行分析。结果表明,各样品的ITS1长度为259~260 bp,ITS2为230 bp,分别有多个特异性信息位点。以ITS序列为依据对石蒜属植物亲缘关系进行分析,表明石蒜属13个种可分为三大类,其中类Ⅰ包括中国石蒜、地笑、安徽石蒜和长筒石蒜,核型为M+T型;类Ⅱ包括矮小石蒜、换锦花、玫瑰石蒜和红蓝石蒜,核型为ST型;类Ⅲ包括稻草石蒜、乳白石蒜、短蕊石蒜和两种人工杂交种,核型为ST+M+T。系统进化树与核型分析结果相似,第Ⅲ类可能为自然杂交种。     

中国石蒜属遗传多样性与亲缘关系的SSR分析

利用SSR-PCR分析中国石蒜属植物的遗传多样性与种间亲缘关系,从46对SSR引物选出18对扩增清晰、多态性好的引物对石蒜属15个种和外类群中国水仙进行了PCR扩增,采用非变性聚丙烯酰胺凝胶电泳和改良的银染法染色,结果表明供试材料具有高度的遗传多样性,多态性位点百分率达97.63%。通过UPGMA法将供试20份材料聚成4类。第I类包括安徽石蒜、长筒石蒜、黄长筒石蒜、忽地笑、中国石蒜、乳白石蒜,其中安徽石蒜与长筒石蒜的亲缘关系很近;第II类包括香石蒜、鹿葱、换锦花、红蓝石蒜和玫瑰石蒜;第III类由石蒜、矮小石蒜、稻草石蒜和江苏石蒜组成;第IV类为外类群中国水仙。分子聚类结果不仅支持乳白石蒜、鹿葱、红蓝石蒜、江苏石蒜、稻草石蒜的杂交起源观点,而且与形态学分类、物种的染色体倍性、核型存在较高的一致性,即除乳白石蒜外,染色体基数接近或等于11、核型含有11t或11t 倍数的物种聚为一大类;染色体基数不等于11、核型为6M+10T的种聚成了另一大类。SSR-PCR容易区分同种不同产地的安徽石蒜或不同花色的长筒石蒜,外形十分相似的石蒜和矮小石蒜,忽地笑和中国石蒜。本研究认为,将安徽石蒜作为一个独立的种不太合适,它可能是长筒石蒜的变种或是以长筒石蒜为亲本的杂交种。     

换锦花和中国石蒜对干旱胁迫的生理响应

以2种春出叶石蒜属植物中国石蒜和换锦花为材料,通过盆栽控水试验,以适宜水分(最大持水量的75%~80%)为对照,设置干旱胁迫(最大持水量的35%~40%)处理,研究干旱胁迫对其幼苗生理生化指标的影响,以明确2种植物的耐旱特性。结果显示:(1)换锦花和中国石蒜幼苗叶片相对含水量(RWC)和叶绿素a、b含量均随着干旱胁迫时间的延长而降低。(2)换锦花可溶性糖含量和脯氨酸含量均随着干旱时间的延长表现出持续增加的趋势,而中国石蒜则表现出先升高后降低的趋势。(3)换锦花和中国石蒜幼苗叶片TBARS含量和相对电导率总体上呈增大趋势,并在干旱末期达到最大值;超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均呈现出先上升后下降趋势。(4)换锦花和中国石蒜幼苗叶片净光合速率(Pn)、胞内二氧化碳浓度(Ci)和蒸腾速率(Tr)随着干旱胁迫时间延长均有不同程度下降。研究表明,在土壤干旱胁迫条件下,换锦花和中国石蒜幼苗叶片在水分生理、光合特性、渗透调节物质和抗氧化酶活性等方面表现出一定的差异,其中换锦花较中国石蒜表现出较强的耐旱性,且具有明显的优势。     

石蒜属种间亲缘关系RAPD分析

采用RAPD标记构建了石蒜属Lycoris植物13个种的指纹图谱。从520个随机引物中筛选出清晰且多态性高的41个引物,共产生了350条DNA片段,分子量在0.3-3.0kh之间,其中253条谱带具有遗传多态性,约占72．3％,平均每个引物扩增的DNA片段数为6．2条。采用TFPGA数据分析软件,计算Nei氏相似性系数和遗传距离,建立了UPGMA聚类图。结果表明：石蒜属13个种明显聚为两大类,即具有单型核型结构（I型）,染色体基数为x=11的5个物种：玫瑰石蒜L.rosea,红蓝石蒜.L.haywardii,稻草石蒜L.straminea,换锦花L.sprengeri和石蒜L.radiata聚为一类;具有两型核型结构（V型和I型）8个物种即江苏石蒜L.houdyshelii,乳白石蒜L.albiflora,中国石蒜L.chinensis,长筒石蒜L.longituba,安徽石蒜Lanhuiensis,夏水仙L.squmigera,短蕊石蒜L.caldwellii和忽地笑L.aurea聚为一类。其中玫瑰石蒜和红蓝石蒜的亲缘关系最近,石蒜和忽地笑的亲缘关系较远,与细胞学的研究结果相吻合。通过RAPD分析,对玫瑰石蒜,红蓝石蒜和稻草石蒜是否天然杂交起源以及乳白石蒜,稻草石蒜和江苏石蒜的分类地位进行了探讨。     

基于叶绿体DNA atpB-rbcL区序列探讨石蒜属种间系统发育关系

摘要：为了探讨石蒜属（Lycoris Herb.）的种间系统发育关系,对石蒜属95个材料包括15种、4变种及2个人工杂种的叶绿体 DNA atpB-rbcL间隔区进行了测序,结合花部形态和核型特征,探讨了石蒜属种间系统关系及其可能的杂交起源,结果表明：在系统发育树上亲缘关系近的材料聚在一起,其中矮小石蒜（L. radiata var. pumila）和换锦花（L. sprengeri）与2个人工杂交种（Hybrid 1、Hybrid 2）、麦秆石蒜（L. straminea）、江苏石蒜（L. houdyshelii）、短蕊石蒜（L. caldwellii）和乳白石蒜（L. albiflora）具有密切的亲缘关系。atpB-rbcL序列揭示的石蒜属种间关系与染色体核型的分类结果部分一致,主要表现在具有近端部着丝粒(A)染色体的种与具有中部(M)和端部(T)着丝粒染色体的种各成一支,与形态和染色体分类结果一致;不同之处在于具有中部、端部和近端部着丝粒染色体的种分散在两个主要分支内,进一步验证了具有中部、端部和近端部3种着丝粒类型染色体组的石蒜如麦秆石蒜、江苏石蒜、短蕊石蒜和乳白石蒜等是杂交起源的假设,结合2个人工杂交种分析,揭示了短蕊石蒜和乳白石蒜的近端部着丝粒染色体来源于换锦花;麦秆石蒜和江苏石蒜近端部着丝粒染色体来源于矮小石蒜。     

石蒜属植物叶微形态特征研究

借助显微和扫描电镜技术对石蒜属植物叶表皮微形态特征进行了研究。利用光学显微镜和扫描电子显微镜观察了石蒜属植物的叶表皮,统计并测量了气孔类型、气孔大小、气孔密度及气孔指数等,描述了气孔及气孔外拱盖的有关特征。结果表明：石蒜属植物叶表皮气孔器为无规则型,近轴面表皮细胞形状、气孔器类型、垂周壁式样、气孔大小及气孔外拱盖内缘种间无差异或极小,表明石蒜属植物为一自然分类群。而远轴面表皮细胞形状、气孔密度、气孔指数、气孔是否下陷、气孔外拱盖是否有蜡质纹饰等种间差异较大。根据远轴面叶表皮细胞形状及叶气孔特征,研究表明：换锦花、长筒石蒜和安徽石蒜,夏水仙、乳白石蒜与红蓝石蒜,石蒜与中国石蒜具有较近的亲缘关系。因此,叶微形态特征对探讨石蒜属植物种间亲缘关系具有一定的意义。     

加兰他敏在忽地笑营养器官中的定位(简报)

石蒜属植物为具有地下鳞茎的多年生草本植物．有重要的药用价值,全属植物约有20余种。我国有石蒜属植物约15种,集中分布于长江中下游地区,野生资源比较丰富。忽地笑（Lycorisaurea Herb．）是石蒜属植物在我国分布较广泛的一种。加兰他敏（Galanthamine）等生物碱是石蒜属植物中主要的药用成分。[第一段]     

Polymorphic microsatellite loci for the genetic analysis of Lycoris radiata (Amaryllidaceae) and cross-amplification in other congeneric species

Lycoris radiata is a perennial herb that has been used in traditional Chinese medicine for a long time and has two main medicinal components in its bulb, lycorine and galanthamine. However, the original microsatellite loci have not been developed for any species of Lycoris. Total genomic DNA was extracted from fresh bulbs using a modified CTAB protocol. We isolated 10 microsatellite loci from 21 L. radiata individuals of a natural population from Yellow Mountain in Anhui Province, China. The number of alleles ranged from two to nine. The observed and expected heterozygosities ranged from 0.238 to 0.952 and from 0.455 to 0.784, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci was characterized in additional five species (L. sprengeri, L. anhuiensis, L. albiflora, L. longituba, and L. chinensis) of Lycoris. The results suggest that these microsatellite markers would contribute to the population genetic studies of L. radiata and other related species.     

安徽石蒜属4种植物核型研究

本文分析了安徽境内石蒜属ＬｙｃｏｒｉｓＨｅｒｂ．４种植物的核型,并结合有关文献探讨它们的核型变异。结果表明,乳白石蒜Ｌ．ａｌｂｉｆｏｌｉａＫｏｉｄｚ．的核型为２ｎ＝１９＝３ｍ＋７ｓｔ＋４ｔ＋５Ｔ,属３Ｂ型;中国石蒜Ｌ．ｃｈｉｎｅｎｓｉｓＴｒａｕｂ．为２ｎ＝１６＝６ｍ＋１０Ｔ,属３Ｂ型;石蒜Ｌ．ｒａｄｉａｔａ（Ｌ’Ｈｅｒ．）Ｈｅｒｂ．为２ｎ＝２２＝４ｓｔ＋１８ｔ,属４Ａ型;换锦花Ｌ．ｓｐｒｅｎｇｅｒｉＣｏｍｅｓ．ｅｘＢａｋｅｒ．为２ｎ＝２２＝２ｓｔ＋２０ｔ,属４Ａ型。以上各种植物均未发现随体和次缢痕,通过核型比较,可见同种不同居群间在染色体数和核型结构上均存在较大的变异。     

换锦花C-带的初步研究

对换锦花C-带进行了初步研究,研究表明其染色体2n=22,带纹主要以着丝点带为主。还讨论了染色体C-带在研究石蒜属植物核型演化及多倍体起源中可能的作用。     

Genetic variations in the chloroplast genome and phylogenetic clustering of Lycoris species

The genus Lycoris of Amaryllidaceae comprises approximately 20 species that are distributed only in the moist warm temperate woodlands of eastern Asia. The objectives of this study were: (1) to clarify the phylogeny of the Lycoris species by using the definitive DNA sequencing method and (2) to examine the possible maternal donor of the hybrid origin Lycoris species and the Japanese triploid strains of Lycoris radiata var. radiata. The nucleotide sequence of the maturase K (matK) gene and the noncoding intergenic spacer (IGS) between the atpB and rbcL genes in the chloroplast genome were determined in a total of 27 strains of 11 species of the genus Lycoris. Variation among taxa was mainly due to nucleotide substitution, although deletions and an insertion were found in the IGS. For two chloroplast regions, the phylogenetic trees showed essentially similar topology, indicating the existence of four clades, I, II, III, and IV. For all the species except L. radiata, intraspecific variation was smaller than interspecific variation. For L. radiata, triploid strains were divided into clades I and II, and diploid strains were divided into clades I and IV. This implies that the diploid species of L. radiata var. pumila is a probable ancestral species. The clustering indicated that the chloroplast genome has not evolved in parallel with the karyotype in genus Lycoris. Regarding the hybrid origin species, the maternal parents of L. squamigara, L. albiflora and L. rosea were revealed to be L. longituba, L. radiata and L. radiata var pumila, respectively. We also suggest that a diploid strain of L. radiata var. pumila in clade I might be a candidate of the maternal donor of the Japanese triploid strains. A possible model of the maternal donor of Lycoris species is proposed.     

两种石蒜生长发育期鳞茎可溶性糖、蛋白质及POD活性的变化

以中国石蒜(Lycoris chinensis)与石蒜(Lycoris radiata)为材料,研究其生长发育期鳞茎可溶性糖、蛋白质含量及POD活性的变化。结果表明,在花芽分化前期,2种石蒜鳞茎可溶性糖、可溶性蛋白质含量均增加,并出现峰值,但从花芽分化后期直到花期结束,可溶性糖含量均持续下降;在花期,2种石蒜鳞茎可溶性蛋白质含量均处于高水平,花谢后明显下降;石蒜展叶期间鳞茎可溶性蛋白质含量明显增加,而中国石蒜花谢后可溶性蛋白质含量处于低水平。在花芽分化前期,2种石蒜鳞茎POD活性呈直线上升,但后期POD活性略有降低;花期后,2种石蒜鳞茎POD活性出现短暂的低谷,随后随着气温的降低而出现一致性上升。     

A Study on Karyotypes of the Genus Lycoris

The genus Lycoris (Amaryllidaceae) consists of about 20 species, all of which are confined to temperate China, Japan and Korea. Cytological investigations, including a reexamination of the karyotypes of 14 taxa, measurements of relative nuclear DNA content, and meiotic configuration observations on some specific forms and interspecific hybrids, have been carried out by the present authors in order to re-evaluate the mode of karyotype evolution and the role of hybridization in the speciation of Lycoris. These have resulted in a new theory for explaining the karyotype evolution in the genus, which will be considered elsewhere. The present paper deals with observations on karyotypes of 11 species, 1 variety and 2 artificial hybrids. Results obtained through karyotype analysis, as shown by the data in Table 1, Plates I-VI and Figs. 1-2, reveal that: (1) the karyotypes of Lycoris rosea, L. radiata var. pumila, L. sprengeri, L. haywardii, L. caldwellii, L. squamigera and L. radiata are, on the whole, consistent with those reported by the previous authors^{[1,2,3,4,5,8,10,12]};(2) the I (rodshaped) chromosomes of L. chinensis and L. longituba are all T’s (telocentric) instead of t’s (acrocentric) or t(Sat)’s; (3) the three materials of L. aurea of different sources have shown a karyotypic differentiation: one with 2n=14=8m+6T, and the others with 2n=16=6m+10T: (4) both of the karyotypes of L. straminea and L. albiflora are 2n=19=3V+6I, inconsistent with 2n=16=6V+10I for the former and with 2n=17=5V+12I for the latter as reported by Inariyama (1953), Bose and Flory (1963) and Kurita (1987). The following aspects are worthwhile discussing: 1. The types of chromosomes. Karyotype analyses reveal the existence of three major chromosome types in Lycoris: (1) m (metacentric) chromosomes: (2) t (acrocentric) chromosomes, with short arms, (3) T (telocentric) chromosomes, sometimes with dot-like terminal centromeres. To distinghish t’s from T’s is of paramount importance for solving the problem of karyotype evolution in Lycoris. Bose (1963) pointed out that in the species with 2n=22, all I chromosomes were t’s, while in species with 2n=12-16, all I chromosomes were T’s. Our results of chromosome observations are consistent with Bose’s remarks. Some authorst^[3,6] have probably mistaken the dot-like terminal centromeres of T’s of L. longituba and L. chinensis as the short arms of t’s. 2. The significance of Robertsonian change in karyotype evolution. Although chromosome numbers and karyotypes are very variable in Lycoris, as shown in Table 1, the total number of arms of a chromosome complement of any species is always multiples of 11. Hence, it seems likely that Robertsonian changes have taken part in karyotype alteration, The genus has a series of basic chromosome numbers: 6, 7, 8 and 11. But which is the most primitive one? It is uncertain whether a successive decrease in chromosome numbers as a result of Robertsonian fusion or a gradual increase in chromosome numbers brought about by fission (fragmentation) has been the essential mechanism for karyotype evolution and speciation in Lycoris. These problems are of crucial importance and will be discussed in our subsequent papers. 3. The origin of polyploids. As evident from Table 1, there are two levels of ploidy differentiation in Lycoris: (1) di ploids with 2n=22 or the equivalent of 22, (2) triploids with 2n=33 or the equivalent of 33. The most common way of origination of triploids in plants is the hybridization of diploids with Tetraploids. But tetraploids have never been found in Lycoris. Thus, it is suggested that the triploids have originated from the combination of an unreduced gamete of a diploid with a normal gamete of another diploid. 4. The role of hybridization in speciation. Results of karyotype analyses show that hybridization has taken an important part in the speciation of Lycoris. Two types of hybrids have been found: (1) 2n=19= 3V+ 16I, L. straminea, L. albiflora and the two artificial hybrids L. sprengeri×L. chinensis and L. haywardii× L. chinensis all possess this karyotype. It could be seen from the above chromosome number and karyotype that this sort of karyotype is exactly half of the total sum of 2n=22I and 2n=16= 6V+10I. It is, therefore, quite evident that taxa possessing this karyotype are all diploid hybrids of 2n=22 and 2n=16, (2) 2n=27=6V+21I, L. caldwellii and L. squamigera possess this karyotype. It is reasonable to assume, too, that they are segmental allotriploids and have arisen from the combination of an unreduced diploid gamete of 2n=16 and a normal haploid gamete of 2n=22. The origin of the hybrid karyotype 2n=17=5V+12I reported by Inari- yama (1953) is similar to that of 2n=19, except that one of the parents possesses 2n=12= 10V+2I instead of 2n=16=6V+10I. The origin of the other hybrid karyotype 2n=30=3V+ 27I reported by Bose (1963) is similar to that of 2n=27, but the diploid gamete comes from taxa possessing 2n=22 instead of 2n=16.