The Study On Rhizosphere Bacterial Of Angelica Based On PCR-RFLP

Angelica sinensis is the perennial herbaceous plants, should belong toumbrelliferae. Is one of the rare traditional Chinese medicinal materials used in ourcountry, it used the root as medicine. It could enrich the blood, promote bloodcirculation, regulate the menstrual function, dryness and slide intestine effectively.The main planting regions are Gansu, Yunnan and Sichuan,et al.Especially theMinxian of Gansu province has cultivated high quality Angelica. Recently, there areserious problems during the period of increasingly cultivating of the crude drugbrought by continuous cropping. Therefore, the first priority is to deal with theobstacles of continuous cropping and to improve the quality and production ofAngelica sinensis. In the experiments described herein, the method of16s rDNAPCR-RFLP is used to complete the comparative study of rhizosphere bacterialcommunity structure and diversity, which is been sampling from the fields ofSoybean-Angelica rotation, Angelica cultivation and wasteland. It’s aim atunderstanding the community structure and diversity of microorganisms in the threesampling sites, as well as providing a theoretical basis for solving Angelica plantationcropping obstacles.In this paper, rhizosphere soil bacterial diversity is deeply studied, which issampled from the fields of Soybean-Angelica rotation, Angelica cultivation andwasteland in Huichuan town of Weiyuan County. Total genomic DNAs of bacteriawere extracted directly from the soil. After purification, the16S rDNAs wereamplified from the total genomic DNA by PCR with the bacterial universal primers799F/1492R. After connecting the amplifier into pMD19-T vector and transforminginto Trans5α, the positive clone was screened by white bacterial colony, and then theclone library was constructed. The16S rDNAs were amplified from the insertedpartial sequence by colony PCR with the universal primer M13of pMD19-T vector.The colony amplifiers were digested by Hae III、 Hha Ⅰand Hinf I. The cleavageproducts will electrophsised on1.5%agarose gels and then the RFLP patterns wereobtained by statistic analysis of gel. The diversity indices were calculated accordingto the RFLP patterns. The results are obtained as follows: (1) The digested results of the bacterial16S rDNA clone library from threesampled field.From the digested band spectrum cluster analysis of each sample, it is easy tofind that there is more bacterial16SrDNA clone library digested type in wastelandsample, which is containing97types of OTU and the total number is253. Besides,there are91types of OTU (total number is351) is found in the rotation field sample,and88types of OTU (total number is286) is found in the Angelica field sample. Theproportion of single clone is the wasteland(21/253)> the Angelica field(13/286)> therotation field(11/351).（2）Theanalyze results of the bacterial population diversity from three sampledfield.From Shannon Diversity Index(H) and abundance index(Schao1, S),whichreflect the diversity of soil bacteria species, the trend we found is the wasteland> therotation field> the Angelica cultivation field. It shows that bacterial species inSoybeans-Angelica rotation field are more richer than that in the Angelica cultivationfield.This further illustrates the Angelica rotation with soybean can help to increasethe diversity of soil bacterial species.The trend of Simpson Index(D) is same to theaforementioned two index. It shows that different cultivation methods may affectbacterial species dominance. In the wasteland, bacterial species dominance is small.But in the angelica cultivation field,bacterial species dominance is obvious. Evennessindex(Species Evenness, E) which reflects the distribution of bacterial species alsoshows the wasteland> the rotation field> the Angelica cultivation field. Obviously,thedistribution of bacterial species in wasteland soil is most uniform. The bacterialcommunities function system is better,too. In the rotation soil, the distribution ofbacteria species is uniform. It also can constitute perfect bacterial communitiesfunctional system. The evenness of bacteria species in Angelica planted soil is smallerthan the aforementioned two plots.(3) The sequenced results of the bacterial16S rDNA clone library from threesampled field.The three sampled field of16S rDNA sequences showed that the sequencedclones fell into ten major lineages within the domain bacteria. Crop rotation and continuous cropping has important influence on Composition and quantity ofPlanctomycetes、SpirocHae tes、Gemmatimonadetes、Firmicutes、Acidobacteria、Actinobacteria、Verrucomicrobia、Armatimonadetes、Chlorobi.