Screening Of Genes Related To Codonopsis Radix Polysaccharide Synthesis And Construction Of Eukaryotic Expression Vector Of Cp1-SST

Codonopsis Radix,the traditional Chinese medicine in China,has been used both as medicine and food.The Midwest of China is its main producing area.It exhibits the functions of strengthening spleen and lung,invigorating spleen-stomach,tonifying MiddleJiao and Qi,and belongs to the traditional tonic Chinese medicine.The bioactive components of C.pilosula include polysaccharides and sterols.Polysaccharides have a wide range of pharmacological effects including anti-tumor,anti-oxidation,regulating body immunity and anti-aging.Polysaccharides are also important indicators for the quality evaluation of C.pilosula.The content of polysaccharides in the authentic medicinal material Lu Dangshen?C.pilosula?in Shanxi Province is 2-4 times higher than that in other production areas.And Lu Dangshen has authentic features such as oily section.It is an important indicator for the quality evaluation of C.pilosula.However,in recent years,due to the unitary germplasm of C.pilosula,the disorderly introduction has led to a serious decline in the quality of cultivated C.pilosula.Therefore,the breeding of excellent C.pilosula based on the polysaccharides among germplasm resources in C.pilosula is necessary.It is of great significance for improving the quality of Lu Dangshen medicine and promoting the high-quality production of medicinal materials to analyze the metabolic pathways of Lu Dangshen polysaccharides at the molecular level,to excavate the key genes of C.pilosula polysaccharides,to regulate the gene expression to achieve precise control of the content of C.pilosula polysaccharides,and to upgrade the genetic improvement of Lu Dangshen germplasm resources.Based on the C.pilosula transcriptome database constructed previously,this study is?1?to analyze two candidate genes CpnsLTP and CpCA that are closely related to the accumulation of polysaccharides in C.pilosula combined with qRT-PCR technology and bioinformatics;?2?to construct the CpCA VIGS silent expression vector and preliminarily optimize the silencing system;?3?to construct the eukaryotic expression vector of the C.pilosula Cp1-SST gene to lay the foundation for further analysis of the polysaccharide metabolism pathway in Lu Dangshen.The main results are as follows:1.Based on the Codonopsis transcriptome database constructed previously,two candidate genes,CpnsLTP and CpCA,positively and negatively related to the accumulation of C.pilosula polysaccharides,respectively,were selected by using Unigene's FPKM values in different tissues and qRT-PCR technology combined with the change trend of polysaccharide content at the early stage of the research group.2.By TA cloning and bioinformatic software,the results showed that the CpnsLTP gene,of 707 bp in length,a 348 bp ORF,with nsLTP specific sites,belongs to the AAI-LTSS super family;CpCA gene is 1186 bp in length,contains a 789 bp ORF,encodes262 amino acids,and the encoded protein has a conserved CA catalytic site.CpCA gene belongs to the CA super family,and the amino acid sequence alignment results showed that it has a higher degree of similarity to other species.Phylogenetic tree showed that CpCA is closely related to the yellow top chrysanthemum CA.3.Using the C.pilosula CpPDS gene as a reporter gene,the VIGS infection system of C.pilosula was established and optimized.The results showed that the photobleaching phenomenon of C.pilosula leaves appeared on 20 days after treatment,if the seedling was treated at 4 weeks old,with infection volume 4 mL and the concentration of the agrobacterium OD₆₀₀ at 1.4,which was earlier than that in the previous result?42 days?,and the testing period was greatly shortened by 22 days.Based on the CpCA sequence,its non-conserved region was cloned,from which the pTRV2-CpCA silencing expression vector was constructed,providing the basis for its function analysis.4.The full-length ORF of Cp1-SST gene was amplified by high-fidelity DNA polymerase.After removing the stop codon,it was integrated into the eukaryotic expression vector pPICZ?C.After it was confirmed that the Cp1-SST eukaryotic expression vector pPICZ?C-Cp1-SST was successfully constructed,it was then transformed into Pichia pastoris X-33 by electroporation with linearized recombinant plasmid.It was then verified that the target gene was successfully integrated with the yeast genome,which will provide the basis for the enzymatic analysis of Cp1-SST gene.