| Anoectochilus,also called ’Bird Ginseng’ and ’Longhairy Antenoron Herb’,is a kind of rare and precious Chinese medical herb,which has a high medicinal value and economic value.In recent years,driven by economic interests and limited resource,wild Anoectochilus has suffered excessive exploitation and excavation,and increasingly scare,and it has already been listed as an endangered medicinal plant by Fujian provincial government.According to the literature,original Anoectochilus mainly locates in Fujian and Taiwan district.Now there are a number of Anoectochilus and its adulterants to sale on the market due to its high pharmaceutical value,which seriously disrupts the status and application of Anoectochilus on the market.Thus,accurate identification Anoectochilus,which is bound to affect the safety and clinical efficacy of TCM medication,becomes more important and even confronts enormous challenges.Currently,the identification of medicine mainly relies on the character and microscopic identification and other traditional methods.With the development of Chinese medicine industry and the increasing amount of identify workload,traditional identification methods have suffered a certain of limitations,which cannot meet the needs of hospital,pharmacy,enterprise and other industry.In this syudy,we established a new rapid and accurate method to identify A.formosanus by DNA barcoding(ITS2 sequences).The main contents and results of experiments are as follows:1.Surveies of Wild A.roxburghii resources include in-suit resource,samples collection of a variety of goods origin and quality evaluation.In this part,we investigated three districts in Fuzhou,Fujian Province where there exists A.roxburghii distributing.Then we analyzed the A.roxburghii species,biological characteristics,environmental characteristics and species which are easily confused with A.roxburghii.This can provide some applied reference for systematic exploitation of A.roxburghii resourses.Survey results shows that:wild A.roxburghii is mainly distributed in altitude between 500 m-700 m,latitude and longitude for Sandiejing(latitude 26 ° 15’41.57 "-26 ° 16 ’10.65",longitude 119 °09 ’43.47 "-119°09’31.69"),Qishan(latitude 25°58’44.56 "-25°58’22.86",longitude 119°07’43.40"-119°07’17.85")and Tianmen(latitude 25°49’07.14 ",longitude 119°00’10.62").The differences of A.roxburghii plant morphology may be influenced by climate,photosynthesis and soil in different regions,and other ecological environment,which may cause mutations of individual.We gathered fresh and dry herbs samples from different commodity markets,pharmacies and companies respectively.34 samples were collected for observation,analysis and species identification.Results shows that Adulterants-varieties confusable class Anoectochilus roburghii on the market mainly are Goodyera repens and Ludisia discolor.2.We used DNA barcoding ITS2 sequences to identify molecule of A.roxburghii and its adulterants.The experiment results shows that there are considerable differences between A.roxburghii and its adulterants in ITS2 sequence;ITS2 secondary structure analysis indicates that there are clearly differences in four stem loops:the numbers of ring,size,location,and the degree of helix.The maximum intraspecific K2P genetic distance in A.roxburghii is much shorter than the adulterants species.It can be seen from the NJ tree construct that A.roxburghii and its adulterants can be clearly separated by DNA barcoding ITS2 sequences.ITS2 sequences of DNA barcodes can quickly and easily identify the A.roxburghii and its adulterants.This provides important molecular basis for gemplasm source identification and the clinical application.3.We separated and purified 17 endophytes from roots,stems and leaves of A.roxburghii in which 8,5,4 were isolated from the roots,stems leaves,respectively.Separation rates of roots,stems,leaves were 46.9%,28.1%,15.6%,respectively.Using traditional morphological identification and molecular characterization of fungal ITS method,the results shows that endogenous fungal classification and identification are 4 classes,6 orders,6 families,8 genera.Penicillium,in which A.roxburghii is the dominant bacteria,account for 29.4%of the isolated total strains;genera Aspergillus and anthrax(17.6 percent respectively of the total strain)takes the second place.What’s more,we tested the antibacterial activity of screened strains.The results shows that FZ1(xx02)、FZ2(xx03)、FZ3(xx06)and FZ11(xx29)inhibited the Colletotriehumeamelliae Masse;FZ2(xx03)、FZ3(xx06)and FZ10(xx28)inhibited the Colletotrichum gloeosporioides;FZ5(xx09)、FZ6(xx 10)、FZ8(xx 18)and FZ12(xx32)were against Staphylococcus aureus.18 kinds of selected endophyte shows no significant inhibition to B.subtilis. |
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