Study On Biological And Genetic Characteristics Of The Endangered Medicinal Plant Atractylodes Lancea(Thunb.) DC.

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.