Transcriptome Analysis And Data Mining Application Research Of The Original Plants Of Fritillariae Cirrhosae Bulbus

Fritillariae Cirrhosae Bulbus is a precious traditional Chinese medicine,the dry bulbs of Fritillaria cirrhosa,Fritillaria unibracteata,Fritillaria delavayi,Fritillaria przewalskii,Fritillaria wabuensis or Fritillaria taipaiensis are the main sources of Fritillariae Cirrhosae Bulbus.It can be divided into “Song bei”,“Qing bei”,“Lu bei”and “Cultivar”,according to the difference of characteristics of Bulbus.The main original plant of “Song bei” is Fritillaria unibracteata;the main original plants of“Qing bei” are Fritillaria cirrhosa,Fritillaria przewalskii and Fritillaria taipaiensis;the main original plant of “Lu bei” is Fritillaria delavayi;Fritillaria wabuensis is the main source of “Cultivar”.Due to the high price and remarkable pesticide effect of Fritillariae Cirrhosae Bulbus,there are lots of fake Fritillariae Cirrhosae Bulbus in the market,which are not as expensive and effective as Fritillariae Cirrhosae Bulbus but have similar morphological characteristics,for the reason of the quality of “Song bei”is better than “Qing bei”,“Lu bei” and “Cultivar”,it is also common to use “Qing bei”or other inferior Fritillariae Cirrhosae Bulbus to impersonate “Song bei”.According to the research,the contents of active components and pharmacological effects of Fritillariae Cirrhosae Bulbus from different sources were also different.These phenomena will seriously affect the clinical curative effect and the reliability of scientific research of Fritillariae Cirrhosae Bulbus,and hinder the industrial development of Fritillariae Cirrhosae Bulbus.The preliminary work of our laboratory also shows that ITS2 DNA barcode and RFLP-PCR can only identify fake Fritillariae Cirrhosae Bulbus,but cannot identify Fritillariae Cirrhosae Bulbus from different sources.Therefore,it is necessary to develop some molecular markers that can quickly and accurately identify each species of Fritillariae Cirrhosae Bulbus from different sources.It is expected to obtain molecular markers for the specific identification method of Fritillariae Cirrhosae Bulbus from different sources and its related species by analyzing the transcriptome data of bulb tissue of Fritillariae Cirrhosae Bulbus of different original plants and ITS1 sequences of different Fritillaria species in this thesis.It has not only guiding significance to the clinical application and scientific research of Fritillariae Cirrhosae Bulbus,but also has great practical significance to the industrial development of Fritillariae Cirrhosae Bulbus.The third generation transcriptome sequencing of bulb tissue of Fritillariae Cirrhosae Bulbus of different original plants was carried out and a total number of225423 non-redundant transcripts Unigene were obtained.There are a total number of12530 Lnc RNA and 132784 complete ORF and 15001 transcription factors were predicted by analyzing the transcriptome data.The largest number of Unigene were annotated into Elaeis guineensis,Phoenix dactylifera and Musa acuminata,according to the results of NR database annotation,indicating that the original plants of Fritillariae Cirrhosae Bulbus are closely related to these species.GO function annotation showed that the most of Unigene was annotated in the Cellular Component category,followed by Biological Process category,and the least annotated in the Molecular Function category.KEGG annotation showed that there are 553 Unigene were involved in terpenoid skeleton biosynthesis pathway,and 404 Unigene were involved in steroid biosynthesis pathway,which provided abundant genetic data for mining and functional identification of alkaloid biosynthesis pathway functional genes in Fritillariae Cirrhosae Bulbus.A SQS ORF c DNA with a full length of 1230 bp,designed as FuSQS was cloned by RT-PCR based on the transcriptome data.The results of bioinformatics analysis showed that FuSQS encoded a protein with 409 amino acids,this protein was a non-hydrophilic protein with two transmembrane domains,and belong to isoprene biosynthesis superfamily I,also had catalytic activity of Farnesyl pyrophosphate.The phylogenetic tree of SQS amino acid sequences of different species showed that FuSQS and Alisma plantago-aquatica,Dendrobium officinale and Zea mays clustered into monocotyledons,this result was consistent with morphological classification result.The q PCR result indicated that the expression level of the FuSQS was different in various tissues.These results laid a foundation for further study on the function of SQS in alkaloid synthesis pathway of Fritillariae Cirrhosae Bulbus.In addition,the similarity between the FuSQS sequence and the ORF sequence of SQS Unigene from transcriptome data was 99.59%,which indicated that the obtained transcriptome data had high accuracy and could be used for screening of specific SSR molecular markers.A total number of 40129 SSR loci were searched,which distributed in 32553 Unigene from the third generation sequencing transcriptome data.The SSR loci included single base repeats to six base repeats,the main repeat type was single base repeats,followed by three base repeats and the number of five base repeats were the least.A total number of 104 pairs of SSR primers were designed with our own specific SSR loci screening conditions and primer screening principles.Finally,we found that No.92 primer could stably amplify the specific band of Fritillaria delavayi at about250 bp,and No.102 primer could stably amplify the specific band of Fritillaria przewalskii at about 170 bp after PCR amplification and electrophoresis.These results indicate that it is feasible to develop specific molecular markers of Fritillariae Cirrhosae Bulbus by using specific SSR loci to design primers,and provide reference for further screening of SSR molecular markers which can be used to quickly and accurately identify Fritillariae Cirrhosae Bulbus from different sources and other related species of Fritillariae Cirrhosae Bulbus.