【作者】 黎开强；

【导师】 吴卫；

【作者基本信息】 四川农业大学 ， 药用植物学， 2009， 硕士

【摘要】 贝母属Fritillaria L.隶属于百合科Liliaceae,为多年生草本植物,全球130种,主要分布在北半球南温带地区。我国产43种和19个变种,其中四川15种4变种,是我国贝母种类集中分布的地区,而且该地区还具有一些保持较多原始性状的种。中药川贝母为川贝母Fritillaria cirrhosa D.Don、暗紫贝母F.unibracteata Hsiao & K.C.Hsia、甘肃贝母F.przewalskii Maxim.& Batal.及梭砂贝母F.delavayi Franch.的干燥鳞茎,具有清热润肺、止咳化痰之功效,为常用中药材之一。在四川省,除广泛分布有《药典》(2005)规定的川贝母药材的四种原植物外,还有许多其他的贝母属植物,多数也在当地作川贝母药材使用。川产贝母属植物多数生长地海拔高,生境独特。截至目前,川贝母药材绝大多数仍依靠采挖野生资源。由于生长缓慢、过量采集和生境恶化等原因,其蕴藏量急剧下降。对其资源进行有效保护以及开展引种驯化迫在眉睫。为此,本文采用细胞学手段和分子标记技术对川产贝母属植物进行遗传多样性分析以及分子鉴定研究。并以其中的川贝母F.cirrhosa为供试材料,探讨了温度和光照对其生长发育、生理指标以及产量和品质等的影响。主要结果如下:1.对川产7种、2变种共10个居群贝母属植物细胞学进行研究。结果表明,其染色体数目均为24条。核型公式如下:川贝母Fritillaria cirrhosa D.DON,2n=24=4m+6st+14t;梭砂贝母F.delavayi Franch.2n=24=2m+2sm+10st+10t;暗紫贝母(折多山居群)F.unibracteata Hsiao & K.C.Hsia,2n=24=4m+6st+14t(SAT);暗紫贝母(茂县居群)F.unibracteata Hsiao & K.C.Hsia,2n=24=2m+2sm+10st+10t(SAT);瓦布贝母Fritillaria wabunesis S.Y.Tang & S.C.Yueh,2n=24=2m+2sm+8st+12t(SAT);甘肃贝母F.przewalskii Maxim.& Batal.2n=24=2m+2sm+6st+14t(SAT);湖北贝母Fritillaria hupehesis Hsiao & K.C.Hsia,2n=24=2m+2sm+12st+8t(SAT);康定贝母Fritillaria cirrhosa D.DON var.ecirrhosa Franch.,2n=24=4m+12st+8t(2SAT);长腺贝母,Fritillaria unibracteata Hsiao & K.C.Hsia var.longinectarea S.Y.Tang & S.C.Yueh var.nov.,2n=24=4m+8st+12t(SAT);短丝贝母Fritillaria Dajinensis S.C.Chen,2n=24=2m+2sm+10st+10t。其中6(变)种染色体核型为首次报道。通过核型分析,认为该属核型向对称性减弱方向发展,推测系统演化上该地理分布的贝母属植物比较原始。2.采用ISSR技术对川产贝母属19份材料进行了遗传多样性分析。结果表明,从35个ISSR引物中共筛选出11个多态性明显、反应稳定的引物,在19份供试材料DNA中共扩增出179条谱带,其中多态性条带163条,占86.8%。材料间ISSR标记遗传相似性系数(GS)在0.569～0.855。聚类分析结果显示,所有供试材料均可区分开,并聚为4组。聚类结果同形态分类有一定的一致性。此外,根据ISSR划分的类群同地理分布有一定关系。《药典》收载的川贝母药材的四种基原植物种间及其与其他川产贝母属各物种间尚不能仅通过采用ISSR分析进行分子鉴定。3.对川产贝母属19份材料进行ITS序列分析。结果表明,所有材料的整个ITS区长度变化范围为658～661bp。其中5.8SrDNA为163bp,ITS1和ITS2长度变幅分别为214～216bp和283～284bp。序列间的遗传分化距离为0.0020～0.0730,平均为0.0270;ITS1和ITS2的进化速率为5.8S的1.2～1.7倍。基于ITS序列分析表明,瓦布贝母和长腺贝母间亲缘关系最近,槽鳞贝母和湖北贝母间亲缘关系次之,槽鳞贝母和川贝母间亲缘关系最远。川产贝母属植物基于ITS序列聚类结果表明,所有供试材料可划分为5类,聚类结果同形态分类有一定的一致性。此外,根据rDNA ITS序列划分的类群同地理分布关系不大。《药典》收载的川贝母药材的四种基原植物种间及其与其他川产贝母属各物种间也不能仅通过采用ITS序列分析进行分子鉴定。4.对川产贝母属19份材料进行matK基因序列分析。结果表明,matK序列长度变幅为1743～1751bp,序列间的遗传分化距离为0.0000～0.0250,平均值为0.0110。其中甘肃贝母和梭砂贝母间亲缘关系最近,浙贝母分别与瓦布贝母和槽鳞贝母间亲缘关系较远。川贝母种质资源基于matK基因序列聚类结果表明,所有供试材料可以划分为4类。聚类结果同形态分类有一定的一致性。此外,根据matK序列划分的类群同地理分布有一定关系。不能仅采用matK序列分析对《药典》收载的川贝母药材的四种基原植物种及其他川产贝母属各物种进行分子鉴定。5.用人工气候箱控制川贝母的生长温度,观察不同温度(15℃、20℃、25℃和30℃)下川贝母的生长发育变化,检测其叶片叶绿素、可溶性糖、丙二醛和脯氨酸含量,并测定鳞茎产量以及总生物碱含量。结果发现,15℃和20℃处理的川贝母生育期较长,四种温度处理下川贝母叶绿素b含量差异均不显著,且15℃、20℃和30℃下川贝母总叶绿素以及叶绿素a含量差异也不显著。此外,川贝母叶片可溶性糖、丙二醛、脯氨酸和鳞茎总生物碱含量,以及鳞茎增长率和干重均随温度降低而增加。据此认为,川贝母不宜生长在过高温度环境,相对较低温度下其生育期较长,鳞茎可适当生长,生物碱含量也有所增加。6.用人工气候箱控制光照强度,在15℃温度下,观察不同光照强度(3000Lx、6000Lx、9000Lx和12000Lx)下川贝母的生长发育变化,检测其叶片叶绿素、可溶性糖、丙二醛和脯氨酸含量,并测定鳞茎产量以及总生物碱含量。结果发现,6000Lx生长发育较好,干物质积累较多,总生物碱含量也较高。光照强度过高或过低,干物质量积累量均大幅度降低。不同光照强度下可溶性糖、丙二醛、脯氨酸含量差异均不显著。6000Lx的光照强度较适宜川贝母的生长。更多还原

【Abstract】 Genus Fritillaria L are perennial herb from Liliaceae. It comprises about 130 species of geophytic occurring in south temperate zone of the Northern Hemispher. There are 43 species and 19 varieties of Fritillaria in china. According to Flora Sichuanica, there are 15 species and 4 varieties of Fritillaria in Sichuan Province, and it is one of the distribution centre of Fritillaria resources in China. Herba Fritillaria recorded in Pharmacopoeia of the People’s Repulic of China (China Pharmacopoeia Committee, 2005), named "Chuan Beimu" in Chinese, are the dry bulbs of F. cirrhosa, F. unibracteata, F. przewalskii and F. delavayi. They are commonly used as antitussive and expectorant medcine. It is one of the common Chinese medicine in china. And some other species (varieties) in genus Fritillaria were also used as Chinese medicine Bulbus Fritiliariae Cirrhosae in Sichuan Province. The natural environments of Fritillaria area from Sichuan Province in high altitude are unique. Currently, Bulbus Fritiliariae Cirrhosae still mainly depends on wild resources. The medicinal plant Fritillaria resources in Sichuan had been badly destroyed because of that retarded growth, excessive collection and worsening habitat. Strengthening environmental protection and introduction and domestication of Fritillaria in Sichuan is imminent. The genetic diversity and the molecular identification were among genus Fritillaria from Sichuan Province were analyzed by using cytology and molecular markers. In addition, this paper also discussed the effect of growth, physiologic indexes, yielding and the contents of alkaloids of F. cirrhosa D. Don under the different temperatures and light intensity. The main results were described as following:1. Cytological studies on ten poputions in seven species and two varieties of Fritillaria from Sichuan, China. The results showed that the chromosome numbers of Fritillaria were 24. The karyological fomula are as follows: F. cirrhosa D. DON, 2n=24=4m+6st+14t; F. delavayi Franch. 2n=24=2m+2sm+10st+10t;F. unibracteata Hsiao & K. C. Hsia(from Zheduoshan poputions), 2n=24=4m+6st+14t(SAT); F. unibracteata Hsiao & K. C. Hsia (from Maoxian poputions), 2n=24=2m+2sm +10st +10t(SAT); Fritillaria wabunesis S. Y. Tang & S. C. Yueh, 2n=24=2m+2sm+8st+12t(SAT); F. przewalskii Maxim. & Batal. 2n=24=2m+2sm+6st+14t(2SAT); F. hupehesis Hsiao & K. C. Hsia, 2n=24=2m+2sm+12st+ 8t(SAT); F. cirrhosa D. DON var. ecirrhosa Franch., 2n=24=4m+12st+8t(2SAT); F. unibracteata Hsiao & K. C. Hsia var. longinectarea S. Y. Tang & S. C. Yueh var. nov. , 2n=24=4m+8st+12t(SAT); F. Dajinensis S. C. Chen, 2n=24=2m+2sm+10st+10t. The karyotypes of the six (varieties) species are reported for the first time. The karyotype of Fritillaria might evolve towords the weakeness of symmetry. It was deduced that Fritillaria populations geographical distrbuted in Sichuan province were more primitive.2. The ISSR marker technique was used to study relationships and genetic polymorphism of nineteen populations in ten species and one varietal species of genus Fritillaria. The results showed that eleven primers were selected from 35 ISSR primers, and 179 DNA fragments were amplified from 19 populations. Of which, 179 fragments were polymorphic (percentage of polymorphic bands was 86.8 %). The genetic similarity among all accessions ranged from 0.569 to 0.855. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 4 groups. ISSR marker technique is suitable for the genetic diversity of Fritillaria from Sichuan Province. In addition, the cluster result of genus Fritillaria had some relationships with the geographical distribution. Interspecific identifications among the four original species of Bulbus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan Province could not be gained by using ISSR markers technique.3. The internal transcribed spacer (ITS) regions of the rDNA were determined in 19 taxon of Fritillaria were cloned and sequenced. Molecular phylogenetic relationships of the related taxon of Fritillaria were studied. The result showed that the sequence of ITS ranged from 658 to 661 bp in length. The 5.8S subunit of Fritillaria was found to consist of 163 or 164 bp nucleotides, while the length of ITS1 and ITS2 varied from 214 to 216 bp and from 283 to 284 bp. The sequences of ITS (ITS1 and ITS2) were about 1.2～1.7 times more variable than those of the 5.8S subunit. The genetic distances varied from 0 .0020 to 0.0730. Based on ITS spacer analysis showed that F. wabunesis had closest relationships with F. unibracteata var. longinectarea, and the relationships between F. sulcisquamosa and F. hupehensis were farther, relatively. F. sulcisquamosa and F. cirrhosa were farthest relatively. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 5 groups. The cluster result of genus Fritillaria and the morphological classification have consistency. In addition, the cluster result of genus Fritillaria had little relationships with the geographical distribution. Interspecific identifications among the four original species of Bulbus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan Province could not be gained by using ITS markers technique.4. The segments of chloroplast DNA (cpDNA), namely matK were determined in 19 taxon of Fritillaria were cloned and sequenced. Molecular phylogenetic relationships of the related taxon of Fritillaria were studied. The result showed that the matK ranged from 1743 to 1751 bp in length. The genetic distances varied from 0.000 0 to 0.025 0, with an average of 0.011 0. Based on matK analysis showed that F. przewalskii had closest relationships with F. delavayi; and the relationships within F. thunbergii and F. sulcisquamosa and F. wabunesis were farther relatively. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 5 groups. The cluster result of genus Fritillaria and the morphological classification have consistency. In addition, the cluster result of genus Fritillaria had little relationships with the geographical distribution. Interspecific identifications among the four original species of Bulbus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan Province could not be gained by using ITS markers technique.5. Using artificial climate controls the growth temperatures(15℃, 20℃, 25℃and 30℃) of F. cirrhosa, the difference of their growth development were observed, the contents of chlorophyll a and b, soluble sugar, MAD, proline of the leavses of F. cirrhosa were tested, and the yielding and the alkaloids contents of the bulbs were analyzed. The growth period of F. cirrhosa under 15℃and 20℃were appropriately extended. The contents of chlorophyll b of F. cirrhosa leaves under four different temperatures are insignificant. The contents of total chlorophyll and chlorophyll a were also insignificantly different under15℃, 20℃and 30℃. The contents of soluble sugar, MAD and proline of F. cirrhosa leaves and the growth ratio, dry weight and content of alkaloids of F. cirrhosa bulb increased with the temperature decreased. Higher temperature is not suitable for the growth of F. cirrhosa. Under the relatively lower temperature, the growth period of F. cirrhosa extended, the bulb can grow properly, and the content of alkaloid increased. F. cirrhosa can improve its cold tolerance by increasing the content of proline and soluble sugar, and it also can maintain the normal content of chlorophyll under the lower temperature.6. The experiment was conducted to study the growth, physiologic indexes, yielding and the contents of alkaloids of F. cirrhosa D. Don under the different light intensity. Using artificial climate controls the growth light intensity (3000 Lx, 6000 Lx, 9000 Lx and 12000 Lx) of F. cirrhosa with temperature of 15℃. The difference of their growth development were observed, the contents of soluble sugar, MAD, proline of the leavses of F. cirrhosa were tested, and the yielding and the alkaloids contents of the bulbs were analyzed. Under the light intensity of 6000 Lx, the growth and development of F. cirrhosa was better, and the dry weight and the alkaloids contents of the bulbs of F. cirrhosa accumulated more. But the dry weight of F. cirrhosa bulb reduced under the lower or higher light intensity. The contents of soluble sugar, MAD, proline of the leavses of F. cirrhosa under four different irradiances are insignificant. The light intensity (6000 Lx) is suitable for the growth of F. cirrhosa.更多还原