【作者】 李琳；

【导师】 谷巍；

【作者基本信息】 南京中医药大学 ， 生药学， 2012， 硕士

【摘要】 茅苍术Atractylodes lancea(Thunb.)DC.为菊科多年生草本植物,其根茎具有燥湿健脾、祛风散寒、明目等功效,为临床常用药之一,是江苏省著名道地药材,被广泛公认为优质的苍术商品,特别是以产于江苏句容茅山一带的质量最优。近年来由于人为因素及其自身的生物学特性如授粉障碍及种群自身恢复能力差等原因,其分布区及种群数量呈现明显的衰退倾向,资源日趋枯竭,被列为省级珍稀保护植物,江苏省4种濒危药用植物之一。鉴于茅苍术资源濒危的现状,为切实有效地保护野生茅苍术资源,促进江苏省这一优良种质资源的保存,本研究拟通过实地考察并借助于扫描电镜技术及RAPD、SCAR、RT-PCR等分子生物学手段,开展濒危药用植物茅苍术生物学和遗传特性研究,为茅苍术这一优良种质资源的保护、GAP生产和可持续利用提供一定的理论和实践基础。论文研究内容和结果主要包括以下几个方面:(1)茅苍术生物学特性研究:对茅苍术生物学特性进行了实地考察,并借助于扫描电镜技术观察其柱头、花粉形态。茅苍术属异花授粉植物,两性花和单性花异株,且单性花均为雌花,两性花常先于单性花开花,大大降低了受精几率,座果率极低;经扫描电镜观察,茅苍术花粉粒呈类球形,赤道面观椭圆形,极面观3裂圆形,具3萌发沟,外壁表面具刺状雕纹,刺渐尖,尖端无网孔,其他部分及周围有网孔。花粉粒平均极轴长59.37±4.3μm,平均赤道轴长48.62±3.5μm,P/E≈1.22;茅苍术两性花及单性花雌蕊柱头表面两侧均具长的、饱满的指状乳突,花粉附着在柱头乳突上;通常茅苍术单性花雌蕊柱头常二裂,而两性花雌蕊柱头开裂不明显。(2)茅苍术花粉活力测定及贮存研究:采用液体培养及染色法筛选茅苍术花粉萌发最适培养基及活力测定方法,并检测不同贮存条件下花粉的活力。结果表明茅苍术花粉在ME3+16%PEG4000+10%蔗糖液体培养基上萌发率最高,达62.1%,而其它三种染色法均不宜用于茅苍术花粉活力的检测;低温可明显延长茅苍术花粉的贮存时间,在-80℃低温下可贮存60 d。花粉是种子植物的雄配子体,在有性繁殖中发挥着重要作用。研究结果为有效解决茅苍术异花授粉困难提供了保障,为茅苍术野生抚育研究提供了理论依据。(3)茅苍术RAPD分析及SCAR鉴定:运用RAPD分子标记方法,从DNA水平上分析道地和非道地茅苍术的遗传变异,筛选了 100个随机引物,从中选取18个多态性强、重复性好的引物进行扩增,共检测到228个位点,110个多态性位点,多态位点比率为48.25%,道地与非道地茅苍术的遗传距离为0.32;同时筛选出2条道地茅苍术中特异的DNA分子片段分别为S45-1030和S1127-651,将筛选到的特异DNA分子片段回收、克隆及测序,其中S45-1030扩增得到的片段序列长度为1030bp,序列与NCBI上向日葵线粒体的转运半胱氨酸、天冬酰胺、酪氨酸的tRNA基因有很高的相似性,相匹配的825bp中有800bp是一致的,由引物S1127-651扩增得到的片段序列长度为651bp,未发现有同源序列;扩增得到的RAPD分子标记成功转化为重复性和特异性更好的SCAR分子标记。研究结果可作为茅苍术道地、非道地茅苍术鉴定的分子标记,并可有效地对正在非道地茅苍术上流失的遗传信息形成有效的保护机制,以促进这一优良种质资源的保存和可持续利用。(4)茅苍术法呢基焦磷酸合酶基因保守区片段克隆及序列分析:运用同源基因的序列设计引物,以茅苍术嫩叶总RNA的cDNA为模板扩增FPPS基因保守区片段,并进行克隆测序。结果得到两个核苷酸序列同源性为87.95%的片段,其长度均为581 bp,分别命名为FPPS1、FPPS2。通过同源序列比对,FPPS1和FPPS2的氨基酸序列与其他植物FPPS氨基酸序列有较高的同源性,推断FPPS1、FPPS2可能是该基因家族中的两个成员;其中,FPPS1的保守区与向日葵、灰白银胶菊、黄花蒿、母菊、人参、檀香的同源性分别为92.4%、91.9%、89.8%、88.6%、84.7%、81.9%;FPPS2 则分别为 86.6%、85.2%、85.2%、84.0%、81.4%、78.8%。本研究首次克隆了 FPPS基因保守区片段并进行生物学性质研究,为茅苍术药效成分生物合成途径及其生成机理研究提供基础。更多还原

【Abstract】 The Atractylodes lancea(Thunb.)DC.is a kind of perennial herb in Compositae;its rhizome,as a commonly used clinical medicine,has effects of dampness and spleen,dispelling wind eyesight and so on.The A.lancea is one of the geo-authentic crude drugs and recognized as good variety in Jiangsu province,especially in Maoshan area.However,in recent years,the distribution area and population decline became apparent tendency because of various human factors and the biological characteristics of the plant,such as the poor ability of recovering and pollination barriers.Facing to the resources exhausted day by day,A.lancea has been included as one of the four kinds endangered medicinal plants in Jiangsu Province.In view of the present situation of A.lancea,this project studies on genetic resources conservation of endangered medicinal plants of A.lancea by scanning electron microscope technology,RAPD,SCAR,RT-PCR and so on.The dissertation mainly consists of the following aspects:1.Research on the biological features of A.lancea(Thunb.)DC.The biological features of A.lancea(Thunb.)DC.were observed by an on-the-spot investigation;the morphologies of stigma and pollen were inspected by scaxming electron microscopy.A.lancea(Thunb.)DC.belongs to the cross-pollination plant;bisexual flowers and unisexual flowers gender different strains.Most of the unisexual flowers are female.In addition,bisexual flowers often bloom preceding unisexual flowers.Therefore,the chances of fertilization are reduced greatly,and the rate of bearing fruits is extremely low.The pollen grains are quasi-spherical,with tricolpate and spinous sculpture.Its average length of polar axis is 59.37±4.3 μm,and the average length of equatorrial axis is 48.62±3.5 μm;P/E ≈1.22,There are plenty of papilla structures covered both of the stigma surface of the bisexual and unisexual flowers.Generally,the cracking of unisexual flowers is obvious,while the cracking of stigma from bisexual flowers is not obvious.2.Determination of pollen viability and study on storageThe optimum culture medium for pollen germination and viability determination methods were screened out by liquid culture and dyeing methods,and then the pollen viabilities in different storage conditions were detected.The results of the study show the optimal culture medium was ME3+16%PEG4000+10%sucrose,in which the pollen germination capacity reached to 62.1%,while the other three dyeing methods can not be applied to detecting the pollen viability of A.lancea.The low storage temperature can significantly prolong the storage time of pollen of A.lancea.At-80℃,pollen viability could be maintained for 60 days.3.RAPD analysis and SCAR markerGenetic diversities between Maoshan and Yingshan A.lancea.were analyzed by using RAPD marker methods.100 random primers were screened,out of which 18 primers that presented high polymorphism and good repeatability were chosen to amplify.As a result,228 loci were detected.Among them,118 loci were polymorphic,presenting a polymorphic rate of 48.25%.The genetic distance between Maoshan and Yingshan A.lancea was 0.32.At the same time,two Maoshan A.lancea specific DNA fragments,namely S45-1030 and S1127-651 were obtained.Fragment S45-1030 was composed of 1030 bp and exhibited high homology to the Helianthus Cys/Asn/Tyr tRNA mitochondrial gene,matching 800/825 bp;a homologous sequence was not found for the S1127-651 in the GenBank database.Furthermore,we successfully transformed the RAPD markers into Sequence Characterized Amplified Regions(SCAR),and the two SCAR markers were highly dependable molecular markers for discriminating the Maoshan A.lancea.from Yingshan A.lancea..This study not only provided genetic insights into the differences in medicinal composition between the Maoshan and Yingshan A.lancea.but also established a reliable molecular methodology to discriminate between the Maoshan and Yingshan populations.4.Cloning and analysis of FPPS gene conserved fragments in A.lanceaThe cDNA,encoding FPPS in A.lancea,was amplified by homology-based cloning with the cDNA of the total RNA of young leaves as the template.The analysis results revealed that the conserved fragments were 581 bp,and the two fragments had been obtained 87.95%identification in nucleotide acid,named as FPPS1 and FPPS2,respectively.It was deduced that they may be members of the FPPS gene family in A.lancea.Sequencing analysis showed that FPPS1 and FPPS2 had high identity with FPPS from other plants.The sequence homology of A.lancea.FPPS1 with Helianthus annuus,Parthenium argentatum,Artemisia annua,Matricaria recutita Panax ginseng and Santalum album were 92.4%,91.9%,89.8%,88.6%,84.7%,81.9%respectively.Furthermore,the sequence homology of A.lancea.FPPS2 were 86.6%,85.2%,85.2%,84.0%,81.4%,78.8%respectively.The cDNA encoding FPPS from A.lancea.cloned and reported for the first time.The work could provide a foundation for exploring the mechanism of sesquiterpene biosynthesis and application to the other medicinal plants.更多还原