Study On Specific PCR Identification Method Of Ophiopogon Japonicus From Sichuan And Atractylodes Macrocephala Koidzs

Ophiopogon japonicus from Sichuan belongs to Ophiopogon japonicus(L.f)KerGawl.,the large Liliaceae.Ophiopogon japonicus from Sichuan is commomly used root as medicine.It is cured for nourish yin,moisten lung,relieve restlessness thrombosis and so forth.Atractylodes macrocephala Koidz belongs to compositae plants,and its dried rhizome has the treatment effects of raising the yang qi,eliminating dampress,promoting urination,hidroschesis,calming fetus.There are many simples of Atractylodes macrocephala Koidzs from different places,which mainly include Zhejiang,Hebei,Pingjiang and Anhui provinces.Different origins of Atractylodes macrocephala Koidzs are so similar that it is difficult to identify the specie using the traditional methods.Our laboratory had obtained one molecular marker of the simple from Sichuan for identification.Our study aims at using the Randomly Amplified Polymorphic DNA(RAPD)technology to screen the molecular marker of Atractylodes macrocephala Koidz.,thereby setting up a new way to identify the specie,and studying the accuracy,sensitivity and applicability of the method.Main conclusions are drawn as follows:1.24 kinds of pant genomic DNA including Ophiopogon japonicases.and Atractylodes macrocephala Koidzs.were isolated using the plant genomic DNA extraction kit.DNA fragment sizes were approximately 23 kb,the purity of DNA determined from the ratio of optical density of 260/280 ratio ranged from 1.790 to 1.932 and DNA concentration ranged from 121 to 541 ?g/mL.2.According to the sequence named CM503,the specific primers of Ophiopogon japonicus from Sichuan named CM1/CM2 were designed.Through the emergence of band at 297 bp,the specific Polymerase Chain Reaction(PCR)method of Ophiopogon japonicus.from Sichuan was built.The studies on the accuracy,sensitivity and applicability of the Specific PCR methods were done.The results showed that the specific PCR method had the advantanges,such as good accuracy,high sensitivity,good reproducibility and applicability.3.There were five molecular markers screened from four kinds of Atractylodes macrocephala Koidzs for identification using RAPD technology,containing one molecular marker of Atractylodes macrocephala Koidz from Hebei and four from Zhejiang.4.Two molecular markers were selected to recycle,clone and sequence.One was the molecular marker of Atractylodes macrocephala Koidz from Hebei,the other one was the molecular marker of simple from Zhejiang.After linked with the T vector,cultured and sequenced,their sequences of gene were obtained.The DNA fragment size of the sample from Hebei was 835 bp,and the sample from Zhejiang was 647 bp.The former was named as HBZ835,the latter was named as ZBZ647.5.According to the sequences,the specific primers of Atractylodes macrocephala Koidz from Zhejiang named ZBZ1/ZBZ2 and Atractylodes macrocephala Koidz from Hebei named HBZ3/HBZ2 were designed,and their specific PCR methods were built.The specific band's size of the sample from Zhejiang was 540 bp,and the sample from Hebei was 778 bp.The studies on the accuracy,sensitivity and applicability of the Specific PCR methods were done.The results showed that the specific PCR method had the advantanges,such as good accuracy,high sensitivity,good reproducibility,good applicability great prospect.