摘要
川贝母(Fritillaria cirrhosa)为百合科贝母属植物,中国《药典》2005版收载,以鳞茎入药,主要功效为清热润肺,化痰止咳。市场需求的快速增长加速了川贝母资源濒危,已成为我国三级保护物种,其生物学保护工作迫在眉睫。本文在川贝母野外资源调查、产地适宜性分析、生理生态学研究、生物条形码鉴定及cDNA文库建立和功能基因挖掘等方面开展了研究,为川贝母资源可持续利用及生物多样性保护奠定了基础,通过对川贝母资源濒危机制的分析提出了保护策略。综合以上研究,得出如下结论:
(1)川贝母主要分布于我国的川西高原,主产于四川甘孜州。采用TCMGIS系统可以科学、准确地分析出与川贝母道地产区最接近的区域,有利于生产布局的合理规划。贝母类药材按照环境因子可以分为西北高原产区、中东部平原和低山产区及东北山区产区,且生物碱种类与贝母在种的水平上与环境密切相关。
(2)首次对川贝母进行了系统的生理生态学研究,结果表明:川贝母未受到明显的环境胁迫,通径分析表明人工引种过程中光照强度、空气相对湿度、空气温度是影响川贝母生长的主要环境因子。通过提高非光化学淬灭,耗散过多吸收的热能是川贝母防止光合机构破坏的主要方式。川贝母生活史各个阶段均对光照敏感,一匹叶和树儿子强光下受到明显的光抑制,灯笼花阶段强光下可以正常生长。不同群落类型对川贝母的光合生理影响差异显著,相对湿度、土壤湿度和二氧化碳浓度是主要影响因子,光合作用速率与土壤中Fe、Cu、Zn和有效N呈显著相关,香柏+杜鹃灌丛适宜进行野生抚育。海拔与川贝母光合速率呈负相关,气孔导度、光合强度和二氧化碳浓度为关键影响因子。
(3)首次在自然条件下人工复制了植物的返祖现象并研究了其发生机制,发现川贝母表型返祖是生境遭到破坏后的一种生态适应对策,有利于其克服非生物胁迫。
(4)首次对贝母属主要药用植物进行了生物条形码鉴定,结果表明单一的候选序列不能完全把贝母属药用植物区分开,但TrnH-psbA+ITS2组合序列可以成功实现彻底鉴定的目标。另外,我们发现贝母属植物系统发育构建、产地适宜性分析、生物碱种类和实验所选条形码候选序列碱基组成存在显著的一致性。
(5)首次对川贝母花期鳞茎构建了cDNA文库,并对其功能基因进行挖掘。在1330个unigene中,我们获得8个与生物碱生物合成途径相关的基因,7个与生长素调节生长发育相关的基因,23个与环境胁迫相关的基因,16个抗病基因,这些基因的发现为进一步研究基因功能及其开发利用、改良川贝母品质、提高人工引种技术提供了依据。另外,有165个EST可能是其它物种尚未报道的新基因。
(6)从研究结果看,川贝母濒危的原因主要为:生境破碎化和人为采挖过度造成的资源破坏;川贝母自身生理因素决定的种群竞争力下降,如川贝母需要4—6年才能进行有性繁殖、衰退型种群年龄结构、自然种子萌发率和幼苗保苗率低、地理分布区域狭窄等。由此我们提出保护对策:一、就地保护,在原生境较好或生态相似性好的地区建立自然保护区或野生抚育区,把药材生产与生态保护有机结合,建立“生产保护”的模式。二、异地保护,开展人工引种和种质资源保护、建立繁育基地(包括种子、器官组织和基因组DNA等),实现川贝母资源的可持续利用。
F.cirrhosa was a perennial species belonging to Fritillary in Lilium.Chinese Pharmacopoeia(2005) accepted F.cirrhosa as the traditional medicine on relieving coughs and eliminating phlegm.The sharp increase in market demand pushed wild plants to the brink of extinction.So conservation on F.cirrhosa stared us in the face.This article did some research work in resources investigation,analysis on the adaptive cultivated area,descriptors and data Standard,ecophysiology,biological identification through DNA barcodes and construction of cDNA libraries,which established bases on sustainable utilization.We put forward protective strategies for F.cirrhosa according to analysis on threatened mechanisms.At last,some conclusions were achieved from our work.
1.F.cirrhosa distributed in Chuanxi altiplano of China and mainly produced in Ganzi state in Sichuan province.The Traditional Chinese medicine adaptability analyzing system(TCMGIS) based on GIS can output the matched areas of climate comparability linked with "daodi" characteristics in F.cirrhosa in scientific and accurate way,which could benefit regulating distribution of cultivated areas.Medicinal Fritillaries were divided into three parts including northwestward produced area,middle and east produced area and northeastward area.Analysis on different species between alkaloid and environmental factors revealed that there was significant relativity.
2.The results of ecophysiological research indicated that F.cirrhosa did not encounter stressful environment.Path analysis showed that photosynthetic photon flux density (PPFD),air relative humidity and air temperature were key ecological factors which greatly effected growth of F.cirrhosa in reintroduction.F.cirrhosa protected photosynthetic reaction centres from damage by increasing non-photochemical quenching to dissipated redundant thermal.Each stage in life history of F.cirrhosa was sensitive to irradiance and only Denglonghua stage could grow well exposed to air. Shade could obviously increase net photosynthetic rate.Communities had a significant influence on physiological characteristics of F.cirrhosa:relative humidity,soil humidity and CO2 concentration were key factors.There was obvious relativity between photosynthetic rate and Fe,Cu,Zn,effective N in soil.Correlation analysis showed negative correlation between altitude and photosynthetic rate and gas conductance, irradiance and CO2 concentration were the most important factors.
3.We firstly copied plant atavistic phenomena in nature and studied its mechanism. Results showed that the atavistic phenotype in F.cirrhosa was a ecological strategy against environment degression,which may help plant overcome abiotic stresses.
4.We firstly used biological barcodes to identify medicinal Fritillaries.Data analysis indicated that single sequence could not distinguish them completely but combinatorial sequences TrnH-psbA+ITS2 could carry out the aim.In addition,we found there were similarities in taxonomy of Fritillary using phylogenetic analysis,analysis on the adaptive cultivated area,alkaloid kinds and gene location variation.
5.Firstly,we constructed cDNA libraries with F.cirrhosa bulbs in flowering stage and analyzed relative functional genes with bioinformatics.We found 1330 unigenes all together which included 8 genes associated with alkaloid biosynthesis,7 genes with growth,23 genes with environment stress,and 16 pathogenesis-related genes.Those genes provided information for studying their functions,improving quality of medicinal materials and improving the level of cultivation.In addition,there were 165 EST undiscovered in other species.
6.We considered that F.cirrhosa was threatened due to fragmentized habitats,excessive wild collection,and its own weak physiological competitive ability such as degressive old makeup,low bourgeoning rate and narrowly distribution.Protective strategies were put forward to carry out sustainable utilization:in situ conservation and ex situ conversation.
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