Transcriptome Analysis Of Oil Degradation During Germination Of Safflower And Several Economic Plant Tissue Cultures

Safflower is a new type of oil crop.The linoleic acid content of safflower oil is as high as 75-85%.It has high medicinal and edible value and can be used to prevent cardiovascular diseases and promote the growth and development of skin and hair.Low safflower seed yield due to geographical and other conditions.Plant tissue culture technology is widely used in the production of crops and traditional Chinese medicine,which can rapidly multiply in a short period of time and obtain more plants.Therefore,a large number of safflower materials were obtained by plant tissue culture techniques,and functional genes related to linoleic acid metabolism were screened by transcriptome analysis,which could lay a foundation for subsequent safflower genetic improvement.At present,the safflower tissue culture system is still unstable,and it is necessary to establish a stable tissue culture system.In addition,the application of plant tissue culture techniques in Chrysosplenium macrophyllum and potato was explored.Chrysosplenium macrophyllum is a scarce wild medicinal plant that needs to be expanded and cultured by tissue culture and rapid propagation;potatoes are widely used,and the growth cycle can be shortened by one-step seedling research.Therefore,this study studied the plant tissue culture on safflower,Chrysosplenium macrophyllum,and potato:1)Using safflower seeds to germinate sterile seedlings on MS medium,7d sterile seedlings were used to induce callus on the callus medium MS+NAA 0.5 mg/L+6-BA 1 mg/L.The dedifferentiation medium was MS+NAA 0.2 mg/L+6-BA 1 mg/L was used to induce seedlings and transferred to MS+IAA 0.05 mg/L to induce seedling rooting.2)The Chrysosplenium macrophyllum adopts the hydroponic rooting method.The experiment shows that it is easier to grow under the condition of clear water+NAA 0.05 mg/L and clear water+IAA 0.05 mg/L,the survival rate of seedlings in transplanted soil after rooting is 100%3)The potato rate was 168%when the potato stem-induced potato was treated with MS+6-BA 2 mg/L+8%sucrose.When the induction condition was MS+IAA0.05mg/L+1%sucrose,the root number was the highest after 10 days of culture,and the root length was more than 10cm.Safflower is called"the king of linoleic acid".Because linoleic acid is unstable and easily oxidized,it is used for medicinal purposes.As a wild relative of safflower,Carthamus lanatus has similar and stable fatty acid composition,and can be used to screen candidate genes for safflower agronomic traits without affecting oil quality.However,there are still few reports on the research of Carthamus lanatus oil,especially the lack of functional genes related to its metabolic pathway.In this study,Xinjiang`s safflower?XJ?,Anhui`s safflower?AH?,and Carthamus lanatus?MH? seeds were germinated for 0 d,1 d,3 d,5 d,7 d,and 10 d,the total of 6 periods were taken for oil content detection The seeds were germinated for 1 d,3 d,5 d,7 d,and 10d,total 5 periods,3 replicates in each period,and 45 samples were analyzed by transcriptome sequencing.On this basis,the dynamic expression regulation process of major functional enzymes during oil degradation was studied.The main results are as follows:1.A method for the determination of linoleic acid oleic acid was established by high performance liquid chromatography?HPLC?.A chromatographic process was set up by using Outstand C18 HPLC Column,a mobile phase of 85%acetonitrile-15% phosphoric acid solution?0.1%?was used with a flow rate at 1.0 mL·min^-1,column temperature at 30?,and determine wavelength at 203 nm.The calibration curve of oleic acid was linear between 0.8722¹7.444 g·L-1,and linoleic acid was linear between 0.1103².205 g·L-1,with the average recovery 99%-100%and RSD of 0%?n=3?.2.The linoleic acid and oleic acid content of seeds of XJ,AH and MH were detected according to the method to 10 d during germination.The linoleic acid content was higher than the oleic acid content in the six periods of three safflowers.At 0-1d,the total oil content increased,the oil content of MH seeds was lower than XJ and AH,and the total oil content during 1d-10d.The degradation rate of XJ and AH was significantly different,and the total oil content of MH was extremely low on the10th day.3.A total of 278.24 Gb data was obtained from transcriptome sequencing.After assembly and de-redundancy,200,111 Unigenes were obtained.The total length,average length,N50,and GC content were 288,404,415 bp,1441 bp,2,132 bp,and41.17%,respectively.A total of 104,697 Unigenes were compared with the KEGG database,and 41,420 Unigenes were annotated with the GO database.A total of84,922 SSRs were detected in 56,912 Unigenes,and 4,868 Unigenes encoding transcription factors were predicted.110821 CDS detected for Unigenes using Transdecoder.4.Using EF-1?as the internal reference gene,analysis the expression patterns of fatty acid synthesis,linoleic acid metabolic,?-oxidation and?-oxidation key enzyme gene in different periods of XJ,AH and MH by real-time PCR.The results showed that there were a lot of differential genes in germination of safflower and Carthamus lanatus.PCR results Consistent with the transcriptome sequencing results,indicating the reliability of the sequencing results.Through the analysis of oil content and transcriptome analysis,it was found that there were significant differences in the content of safflower and Carthamus lanatus.There were also a large number of differential genes in the lipid metabolism pathway,In the?-oxidation pathway,the differential gene expression was lower in the Carthamus lanatus,but in the?-oxidation pathway,the differential gene was expressed in the Carthamus lanatus.FAD2 has a low expression level on the first day of germination of the geranium seed,which may be replaced by other homologous genes.The expression of linoleic acid metabolic pathway degradation gene gradually increased,lipoxygenase?LOX2S?and linoleic acid 9S-Lipoxygenase?LOX1?is expressed in the Carthamus lanatus,which may be due to lower linoleic acid content or other pathways.