| Polygonatum cyrtonema Hua is a perennial medicinal herb of Lilyaceae?Polygonatum Mill?,which combines medicinal,edible,cosmetic and ornamental values,and has broad prospects for development and utilization.Rhizome of Polygonatum cyrtonema Hua contains polysaccharides,flavonoids,sputum,steroidal saponins,lignans and other compounds,and a variety of substances useful for human body.It was flat and sweet,and is a nourishing top grade.It has the functions of nourishing moisturizing,lowering blood fat,lowering blood sugar,lowering blood pressure,improving human immunity,supplementing vital energy,supplementing breath and blood,etc.However,due to the long growth cycle of the Polygonatum cyrtonema Hua,the seed reproduction takes more than 5 years,and the rhizome reproduction takes at least 3 years.The traditional asexual breeding technique not only makes the fine traits of the Polygonatum cyrtonema Hua difficult to preserve,but also has low reproductive efficiency,which greatly limits the scale of the Polygonatum cyrtonema Hua.In recent years,with the increasing demand for Polygonatum cyrtonema Hua,wild resources were far from meeting the needs of production,and factory production was imperative.Plant tissue culture technology can not only preserve the excellent traits of Polygonatum cyrtonema Hua well,but also eliminate the influence of external factors such as seasons,so as to significantly increase the proliferation coefficient in a controlled environment.Therefore,this study used Polygonatum cyrtonema Hua as a material to establish a sterile system for Polygonatum cyrtonema Hua,and induced the growth of adventitious buds,optimized the in vitro culture conditions,and then determined the saponins of the optimized materials,At the same time,the cDNA and gDNA full-length sequences of PcCIGR and PcSCL21 genes were cloned,and the growth of PcCIGR and PcSCL21 genes under different treatments was analyzed.In order to provide reference and reference for in vitro culture and industrial production of Polygonatum cyrtonema Hua.The main Research results were as follows:1 Establishment of Polygonatum cyrtonema Hua sterile systemAn aseptic tissue culture procedure was established for wild Polygonatum cyrtonema Hua in Samming by optimizing the disinfection and the adventitious bud induction procedures,in which the tubers from wild-growing plants were used as explants.Results showed that the best sterilization procedure for explants was as follows:brushing soils off the tubers in clear water with a soft toothbrush;cleaning within diluted detergent for 16 hours;soaking in 3%carbendazim for 2 days?shaking intermittently?and washing in water to remove residual carbendazim from the surface;drying on filter paper for 5 h;dipping first in 75%alcohol for 30 s and then in 0.2%mercuric chloride for 15 min,which resulted in a contamination rate of only 20.2%.Comparatively,explants from March to April of the spring season were significantly better than those from November to December in both the growth and the induction of adventitious buds.It was found that the optimal medium for adventitious bud induction of the wild Polygonatum cyrtonema Hua explants was:MS+6-BA 4.0 mg/L+NAA 0.2 mg/L,and the budding rate on the medium was as high as 88.0%.2 Optimization of in vitro culture conditions of Polygonatum cyrtonema HuaThe rhizome of Polygonatum cyrtonema Hua was used as the material to study the effects of 6-BA,NAA,culture days,light quality and photoperiod on the proliferation of adventitious buds of Polygonatum cyrtonema Hua by single factor experiment.The results showed that the optimal adventitious bud proliferation medium of Polygonatum cyrtonema Hua was:MS+4.0 mg/L 6-BA+0.4 mg/L NAA+30 g/L sucrose+7 g/L agar,and the experimental factors for its adventitious bud proliferationThedegreeofinfluencewas:hormone>photoquality>photoperiod>culture days.The results showed that hormones had significant effects on the proliferation of adventitious buds of Polygonatum cyrtonema Hua,while the days of culture,light quality and photoperiod had little effect on the proliferation of adventitious buds.However,it may have a certain impact on the accumulation of metabolites.3 Extraction and determination of Polygonatum cyrtonema Hua dioscinThe rhizome of Polygonatum cyrtonema Hua was used as the material to extraction and determination of dioscin from processed samples of photoperiod,photoperiod and hormones.The liquid phase separation was performed using ODSC188 column 4.6×150 mm,mobile phase was acetonitrile:water,flow rate 1.0 mL/min,column temperature30?,and injection volume 10.0?L.The gradient elution conditions were starting from 0-5 min to 60:40,7-12 min at 90:10,and returning to 60:40after 12 min.The quantitative wavelength was 210nm.Dioscin was isolated within 5 min.The results of orthogonal test showed that the extraction efficiency was the highest with 75%ethanol extraction,the ratio of material to liquid was 1:20,and extracted for 4 hours.The above method was used to extract and measure the rhizome samples after the relevant treatment,and the results showed that the content of dioscin was the highest when the light quality was white light.The photoperiod that is most conducive to the accumulation of dioscin is 12h/d.When the concentration of cytokinin 6-BA was 4.0 mg/L and the concentration of auxin NAA was 0.2 mg/L,the content of dioscin in rhizome of Polygonatum cyrtonema Hua was the highest and was significantly higher than that of the control group.The content of dioscin also increased with the increase of the number of culture days.4 Cloning and bioinformatics analysis of the genes related to growth and development of CIGR and SCL21 of Polygonatum cyrtonema HuaTo study the mechanism of CIGR?Chitin-inducible gibberellin-responsive?and SCL21?Scarecrow-Like 21?gene under different treatments and different tissues in Polygonatum cyrtonema Hua.Using full-length verification to obtain the CIGR and SCL21 gene's cDNA and gDNA sequence of Polygonatum cyrtonema Hua,then bioinformatics analysis of CIGR and SCL21 gene.The results showed that the cDNA sequence and the full length of gDNA of CIGR and SCL21gene were identical,and the complete open reading frame?ORF?was1770 bp and1569 bp in length,which encoded a total of 589 and 522amino acids.The results showed that the CIGR and SCL21 had no intron structure.Bioinformatics analysis indicated that CIGR and SCL21 protein has a GRAS conserved,which belongs to the GRAS family that was a plant-specific transcription factor.Besides,CIGR and SCL21was a basic protein with no signal peptide;Motif analysis found that CIGR and SCL21 protein was relatively conserved among different species,and the conserved region was mainly located at the middle to the 3'end;Evolution analysis of CIGR and SCL21 amino acid sequence showed that the CIGR and SCL21 proteins in Polygonatum cyrtonema Hua and the Asparagus officinalis maintained a strong genetic relationship.Further subcellular localization revealed that the CIGR and SCL21 protein was localized in the nucleus,consistented with the predicted results.5 Expression Pattern Analysis of CIGR and SCL21 Gene in Polygonatum cyrtonema HuaSelecting sterile seedlings with better growing growth and better growth as test materials.The 18S rRNA was used as the internal reference gene,and the Roche LightCycler 480 instrument was used for the test.The expression of CIGR?SCL21 gene in different tissue parts,culture days and different hormone treatments was detected by qPCR.Real-time quantitative PCR?qPCR?results showed that the CIGR and SCL21gene of Polygonatum cyrtonema Hua had organ expression specificity,and the highest expression in leaves.The expression pattern of PcCIGR and PcSCL21 gene under the conditions of light quality,photoperiod,hormone and culture days was analyzed.The results showed that PcCIGR gene expressed higher under blue light and low expression under far red light,and the expression peak appeared in the treatment of 9 h/d photoperiod.PcSCL21 was most beneficial to the growth of Polygonatum cyrtonema Hua in blue and green light,and the highest expression was observed at the photoperiod of 18h/d.The expression of PcCIGR and PcSCL21 under hormone treatment showed a significant positive correlation with the content of diosgenin,PcSCL21 peaked at a concentration of 0.2 mg/L for NAA and 4.0 mg/L for 6-BA,and reached the highest level at 50 days of culture.This study showed that PcCIGR and PcSCL21 gene could respond to light quality?photoperiod and hormone treatment,and it was speculated that blue light?short daylight and hormone may be more suitable for the growth of Polygonatum cyrtonema Hua. |
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