SNP Markers And RgURT RgTAT Rg4CL RgC4H And RgC3H1 Of Rehmannia Glutinosa Libosch

Rehmannia,Scrophulariaceae,includes six species such as Rehmannia glutinosa,Rehmannia piasezkii,Rehmannia elata,Rehmannia henryi,Rehmannia chingii and Rehmannia solanifolia.Rehmannia glutinosa is mainly distributed in China,Japan and Korea,and its root can be used as medicine.In China,Rehmannia glutinosa Libosch is mainly distributed in Henan,Shanxi,Shaanxi and other provinces.Rehmannia glutinosa planted in Huai County is known as one of the famous "four great medicine",which has a wide medical market.Rehmannia glutinosa has many cultivated varieties,such as wen 85-5,Jinjiu,Beijing NO.3,Hongshuwang,Wenhuai and so on,as well as a variety of wild resources.In the production of Rehmannia glutinosa,due to the mixture of varieties and long-term vegetative reproduction,there are many problems in the process of planting,such as large amount of consumables,virus infection,variety degradation,etc.,so it is necessary to dig effective molecular markers to identify Rehmannia glutinosa varieties,clone genes,verify and synthesize genes by molecular biological technology.In this study,SNP loci were mined in the transcriptome database of Rehmannia glutinosa,and SNP molecular markers were developed by using polymorphic SNP loci to effectively identify Rehmannia varieties.The URT gene was cloned by homologous cloning and electronic cloning,and its prokaryotic expression vector and E.coli engineering bacteria were constructed for prokaryotic expression.On the other hand,the overexpression vector and Agrobacterium engineering bacteria were constructed,and the RgTAT,Rg4 CL,RgC4 H,RgC3H1 genes related to verbascoside biosynthesis which we cloned earlier were used to study the genetic transformation of Arabidopsis and Rehmannia glutinosa.The main results are as follows:1.Using the Rehmannia glutinosa transcriptome data and others databases(SRA databases)in NCBI to develop Rehmannia glutinosa SNPs,a total of 35,339 SNP loci were found in Unigene.Among them,the frequency of base transition was significantly higher than that of base transversion,and the frequency of base transition was about twice that of transversion.For base transition,the highest frequency was 32.33%of A/G and 31.95% of C/T.For base transversion,the highest frequency was 9.36% of A/C.2.According to those forty SNP loci randomly selected by us,39 primer pairs for PCR validation were designed.Through PCR amplification and sequence alignment,7 pairs of specific SNP primers were screened,including 8 polymorphic SNP loci.Fingerprints were constructed based on the differences of 8SNP loci and it was found that seventeen germplasms could be distinguished from twenty-eight Rehmannia germplasms,which complemented the genetic diversity of Rehmannia glutinosa.3.The RgURT gene of was successfully cloned by homologous cloning and electronic cloning.The ORF of RgURT gene is 1413 bp,encoding 471 amino acids.Bioinformatics analysis showed that its molecular weight was 53 k Da,isoelectric point was 5.79,RgURT protein had no signal peptide,subcellular was located in chloroplast,there was a certain transmembrane helix region.RgURT protein belongs to the GT-B superfamily of glycosyltransferase,which has a conserved structure of UDP glycosyltransferase.Multiple sequence alignment of nucleotide and amino acid sequences of the three URT genes extracted from the whole transcriptome sequencing of Rehmannia glutinosa showed that the highest homology between them was 51% and 41%.The recombinant protein GST-RgURT was induced by E.coli system,the plant expression vector pCAMBIA1300-RgURT was constructed,and the expression vectors pCAMBIA1300-RgTAT,pCAMBIA1300-Rg4 CL,and pCAMBIA1300-RgC4 H were transformed into the sensitive cells of GV3101 by freeze-thaw method Agrobacterium engineering bacteria.4.Through agrobacterium mediated transformation,pCAMBIA1300-RgURT,pCAMBIA1300-RgTAT,pCAMBIA1300-Rg4 CL,pCAMBIA1300-RgC4 H and pCAMBIA1300-RgC3H1 were respectively transformed into 85-5 and Jinjiu Rehmannia glutinosa.Five kinds of transgenic resistant calli were obtained through hygromycin resistance screening.Through induction culture,transgenic regenerated plants were obtained and tested at DNA and RNA levels.5.Through agrobacterium mediated transformation,the positive transgenic Arabidopsis plants were obtained by transforming pCAMBIA1300-RgURT,pCAMBIA1300-RgTAT,pCAMBIA1300-Rg4 CL,pCAMBIA1300-RgC4 H and pCAMBIA1300-RgC3H1 into wild type Arabidopsis.Four transgenic lines with RgTAT gene and three transgenic lines with Rg4 CL gene were obtained.In summary,our results developed SNP markers of Rehmannia glutinosa,established its technical system for germplasm identification;expanded the data of Rehmannia glutinosa genes,and established a gene engineering technology system to study the function of genes related to verbascoside biosynthetic.These results will clarified the molecular mechanism of verbascoside biosynthetic,and is the basis for the quality improvement of Rehmannia glutinosa and the production of verbascoside biosynthesis.