Effect Of Codonopsis Pilosula Cp1-SST Gene Overexpression On Tobacco Cold Tolerance And Protein Purification

Lu Dangshen is a genuine medicinal material of Shanxi Province.It tonifies the spleen and replenishes qi,nourishes blood and nourishes jin."According to ancient wood and grass clouds,ginseng is the best for the Party",according to the "Materia Medica Xin".But the formation mechanism of Lu Dang’s reference is not clear.Genuine medicinal materials have the effect of adversity,and their active components are induced by environmental stress to accumulate.Codonopsis polysaccharide is an important functional component and biomarker of Codonopsis.Previous research by the research group showed that fructans in Codonopsis polysaccharide took up more than 50%,which not only has the effect of anti-gastric ulcer,but also can respond to external stress and improve the stress resistance of plants.The key enzyme of fructan synthesis,sucrose 1-fructose transferase(Cp1-SST)gene,was cloned by our research group in the early stage.It was found that after the silencing of Cp1-SST gene,polysaccharide content and fructan content of C.codonopsis were significantly reduced,indicating that this gene was involved in polysaccharide synthesis of C.codonopsis.However,the role of Cp1-SST gene in the stress resistance of Luodonopsis and its protein function are still unclear.Fructan is not contained in tobacco,so it can be used as a natural model plant to study the effect of fructan metabolism.This study based on the early stage of the gene silencing,on the basis of the purpose Cp1-SST gene into prokaryotic expression vector pET28a(+),through the heterologous expression for protein purification purposes,and by using the semal reforming process of agrobacterium mediated the Cp1-SST gene into tobacco,make its excess expression,analysis of transformation of plant and controlled plant morphological differences,and through the low temperature stress processing,the analysis of the effect of overexpression of tobacco resistance gene,for further study of codonopsis pilosula polysaccharide synthesis mechani.The main findings are as follows:1.The full-length Cp1-SST gene sequence was amplified and connected to the protein prokaryotic expression vector pET28 a.After double enzyme digestion and sequencing,the results showed that the protein structural bases were free of mutations and deletions.The pET28a-1-SST prokaryotic expression was successfully constructed.Transform E.coli BL21,induce expression by IPTG,and the protein Marker indicated that there is a single bright band at the relative molecular weight of66.2 KD.The result is consistent with the Cp1-SST protein quality predicted by the research group in the early stage of bioinformatics.Through the optimization of the induction expression system,it was found that the optimal conditions were: the induction concentration of IPTG was 0.1 mM,the induction temperature was 18℃,and the induction time was 16 h.On this basis,the purified Cp1-SST protein was obtained,which laid the foundation for later enzymatic function analysis.2.Using Codonopsis pilosula(Franch.)Nannf root system as material,clone the full-length Cp1-SST gene sequence and connect it to pCAMBIA1381-35 S plant expression vector by seamless cloning to construct pCAMBIA1381-35S-1-SST recombinant expression vector.And introduced into Agrobacterium GV3101.Agrobacterium-mediated tobacco leaf disc genetic transformation method was used to screen and cultivate transgenic tobacco plants.Through DNA-PCR detection,cDNA-PCR detection,and GUS staining analysis,the tobacco transgenic plants(35S::1-SST)were further identified.3.Morphological observations of 35S::1-SST,35S::,WT tobacco plants found that 35S::1-SST tobacco plants grew slower than 35S::,WT tobacco plants,and their plant height and root length were measured.The difference analysis showed that the plant height and root length of 35S::1-SST tobacco were significantly lower than those of 35S:: and WT tobacco,which was statistically significant(p<0.05).It shows that overexpression of Cp1-SST gene has a certain inhibitory effect on the growth and development of tobacco.After 4℃ low temperature stress treatment,35S:: plants have serious albinism,and the leaves have wilting.Analysis of resistance-related indicators found that the chlorophyll decline of 35S::1-SST tobacco was significantly less than the decline of 35S::tobacco chlorophyll;the increase of 35S::1-SST tobacco MDA was significantly less than that of 35S::tobacco MDA.All these results demonstrated that overexpression of the Codonopsis pilosula Cp1-SST gene can increase its resistance to low temperature.