Full-length Transcriptome,RgURT Gene Editing And Metabonomic Analysis Of Rehmannia Glutinosa Libosch

Rehmannia glutinosa Libosch,a perennial herb in the genus Rehmannia of Scrophulariaceae,is a commonly used bulk medicinal material.Rehmannia glutinosa（R.glutinosa）is widely produced in China,mainly distributed in Henan,Shaanxi and Shanxi provinces,among which R.glutinosa produced in Wen county,Wuzhi and Boai counties of Henan Province has the best quality.R.glutinosa,which is used as medicine by root tuber,has many medicinal values such as anti-cardiovascular diseases,enhancing immunity and anti-hypertension,and its efficacy is closely related to its chemical components.As one of the effective components of R.glutinosa,verbascoside has pharmacological activities such as anti-inflammatory,anti-oxidation and anti-tumor.However,as a non-model plant,the full-length transcriptome database has not yet been established,and the key enzymes and their corresponding genes in some downstream biosynthetic pathway of verbascoside of R.glutinosa have not been excavated.In addition,the hybrid and degradation of R.glutinosa varieties affect the yield and quality of R.glutinosa,so it is necessary to explore effective methods of variety identification and find wild variety resources with beneficial traits to solve this problem.In this study,R.glutinosa,R.glutinosa treated with naphthalene acetic acid and wild R.glutinosa were used as materials.The full-length transcriptome sequencing technology was used to establish a R.glutinosa transcriptome database,and the key enzyme genes downstream of verbascoside biosynthesis were mined;CRISPR/Cas9 technology was used to analyze the function of UDP-rhamnose: rhamnose transferase gene（Rg URT）and the editing technology system of R.glutinosa gene was established;Widely targeted metabonomics technology was used to study the differences of chemical constituents between cultivated and wild R.glutinosa.The main results are as follows:1.The full-length transcriptome of R.glutinosa ‘Jiu Jiu’ was sequenced based on SMRT technology and Pac Bio Sequel high-throughput sequencing platform.26.61 Gb of data,96,548 high-quality consistent sequences,61,262 non-redundant transcript sequences were obtained,3,070 alternative splices,34,013 SSR loci,4,241 lnc RNA,46,412 complete CDS sequences and 5,562 transcription factors were identified.And 56,633 transcripts were annotated to 8 databases including NR,Swissprot,GO,COG,Pfam,KOG,egg NOG and KEGG.Based on the annotation results of KEGG metabolic pathways,17 secondary metabolic pathways involving 1,042 transcripts in the biosynthesis of flavonoids,alkaloids,and others were explored,and 326 transcripts（encoding 14 enzymes）were annotated into the verbascoside synthesis pathway,and 401 transcripts（encoding 25 enzymes）were annotated into the catalpol synthesis pathway.Among them,there were four Rg URT genes（gene registration numbers: MN646769,MT767427,MT767428 and MT767429）,18 hydroxycinnamyl acyltransferase genes（HCT）and one Caffeoy-Co A:alcohol caffeoyl transferase gene（CCCT）with the activity of transferring caffeoyl in the key enzyme genes downstream of the candidate verbascoside synthesis pathway.2.Under the same concentration and different induction time of methyl jasmonate（Me JA）solution,RT-q PCR revealed that URT,CCCT and HCT,the key enzyme genes of the downstream pathway of verbascoside biosynthesis,showed different response levels.The results showed that the expression of URT,CCCT and HCT showed a V-shaped change trend,and the overall expression trend remained consistent,which laid a foundation for analyzing the functions of URT,CCCT and HCT genes.3.The content of verbascoside in wild R.glutinosa was 0.2433% and that of R.glutinosa variety‘Qinhuai’ was 0.0101% determined by HPLC.The relative expression levels of URT,CCCT and HCT genes in these two varieties were detected by RT-q PCR.The results showed that the expression levels of different genes in different varieties and the same gene in different varieties were different.Compared with the expression levels in ‘Qinhuai’ R.glutinosa,the expression levels of URT,CCCT and HCT genes in wild R.glutinosa were higher than those in ‘Qinhuai’ R.glutinosa.4.According to the above one Rg URT gene sequence,two target sites were selected,two sg RNAs were designed,and the CRISPR/Cas9 gene editing vector p BWA（V）HS-zmpl-URT was successfully constructed,which was further transformed into Agrobacterium and positive Agrobacterium tumefaciens engineering bacteria were obtained by enzyme digestion and PCR detection.The Agrobacterium transformation method was used to transform R.glutinosa,and 122 candidate gene-edited R.glutinosa seedlings were obtained,43 hygromycin-resistant and Cas9 gene-positive seedlings were screened out;Their genomes were extracted as templates,and two editing sites in Rg URT gene were amplified by PCR.Electrophoresis showed that 43 seedlings all amplified 325 bp and 450 bp target bands.The gel recovery and sequencing results showed that compared with the sequence of the control gene,43 seedlings all showed the-3 and-4 base changes of the target PAM sequence,all of which were changed from AA to TT,and the gene editing rate was 26.23%;20 gene editing seedlings were randomly selected for RT-q PCR,and the expression of Rg URT gene in 12 plants decreased significantly;The results of HPLC showed that the contents of verbascoside in 3 gene editing seedlings were significantly lower;Transplanting gene-edited R.glutinosa seedlings suffered insect pests after two months of cultivation in the greenhouse,showing white spots on the leaves,and the results of stereoscopic microscope observation showed that the leaves were damaged by tetranychus cinnbarinus.5.In this study,the cultivated variety of R.glutinosa ‘ Beijing No.3’ and wild R.glutinosa were taken as the research objects,and the differences of chemical composition between them were studied by widely targeted metabonomics technology based on UPLC-MS/MS detection platform and combined with HMDB,MWDB and METLIN databases.228 secondary metabolites were preliminarily detected,and 58 metabolites with significant differences were identified,among which flavonoids such as flavonoids,flavonols and anthocyanins were dominant in the secondary metabolites of R.glutinosa.The contents of 40 metabolites,such as Protocatechuic aldehyde,Benzoic acid,Robinin,Kaempferitrin and Phytolaccagenin,were significantly different between wild R.glutinosa and ‘Beijing No.3’,which provided an important basis for screening,identification and quality evaluation of iconic chemical components of R.glutinosa.In summary,we have established a full-length transcriptome data set of R.glutinosa,which enriched the existing database of R.glutinosa and provided rich gene sequence resources for molecular biology and molecular breeding research of R.glutinosa.Some downstream related enzyme genes for verbascoside biosynthesis were excavated and verified,which complemented and improved the pathway and molecular mechanism for verbascoside biosynthesis in R.glutinosa.The gene editing system of R.glutinosa was preliminarily established,which laid a foundation for the study of gene function,regulation,genetic breeding of R.glutinosa by gene editing technology.The establishment of the widely targeted metabolomics identification technique for cultivated and wild R.glutinosa has provided a new means for the identification of different varieties of R.glutinosa,chemical component analysis and exploration of quality control indicators.