Studies On The Mechanism Of Carotenoid Synthesis In Petals Of Osmanthus Fragrans Lour. And Embryogenic Callus Culture And Transformation

Osmanthus fragrans Lour.belonging tothe genera Osmanthus of family Oleaceae,is one of China’s traditional ten famous flowers.Osmanthus fragrans are popular because of their beautiful tree shape and unique fragrance,and are widely used in landscape construction.Because of the different flower color and flowering stage,Osmanthus fragrans is divided into four different variety groups:Albus Group,Luteus Group,Aurantiacus Group and Asiaticus Group.Among them,the color of Aurantiacus Group is the darkest orange-orange red,and the color of Albus Group is the brightest white-white yellow.Recent studies have shown that the color of petals of Osmanthus fragrans is closely related to the species and content of carotenoids in petals,but the color fraction of petals of Osmanthus fragrans is closely related to the content of carotenoids.The study of sub-mechanism is less.In this experiment,2-（4-chlorophenylthio）triethylamine hydrochloride（CPTA）was used to treat the flower branches of Osmanthus fragrans,and the results showed that the color of petals changed obviously.Determination of carotenoid and quantitative detection of key genes of carotenoid metabolism pathway were carried out in chromotropic petals,and the regulation factors related to carotenoid metabolism were analyzed in combination with transcript data.At the same time,on the basis of the preliminary work of the research group,the embryogenic calli induction and subculture medium of Osmanthus fragrans were optimized,and the transformation conditions of Agrobacterium tumefaciens were optimized.The main findings are as follows:1.Optimization of anti-browning conditions in primary and subculture mediaAccording to Zou Jingjing（2014）classification standard of Osmanthus fragrans fruit,the zygotic embryo of Osmanthus fragrans in stage 3 was selected as material.Then used as the material to design the embryogenic calli induction medium with different auxin and mitogen combinations.It was found that the induction rate of embryogenic calli in the concentration of 0.5mg/L NAA 1.0mg/L KT was 73%,and the browning rate was only 3%in the later stage of culture.The photoperiod and basic medium types in the subculture medium were designed.The results showed that the embryogenicity of embryogenic calli could be maintained under dark conditions,while the embryogenic calli were loose and browning rate decreased with the change of MT medium.At the same time,add to the subculture medium.2.Optimization of Agrobacterium tumefaciens Transformation conditionsThe induced embryogenic calli were infected by Agrobacterium tumefaciens,and the selective sensitive concentration of kanamycin was in the range of 100-150mg/L.The sensitive concentration of Cefalexin was within the range of 200-300mg/L.The results of transient expression of Gus showed that when Agrobacterium tumefaciens was EHA105,the concentration of AS was 200 mg/L,and the incubation time was 1day,the transient infection efficiency was higher than that of Agrobacterium tumefaciens.When the concentration of infecting bacteria solution OD₆₀₀was 0.6 and the days of co-culture was 6 days,the rate of returning bacteria was low and the instantaneous infection efficiency was higher.Adding 150mg/L Km 200mg to screening medium.3.HPLC Analysis of carotenoid content after CPTA treatmentAfter soaking in 0.2g/L and 0.4g/L CPTA solution for 24 hours,the color of petals near the liquid surface changed,and after 36 hours of immersion,the color changed obviously.The results of carotenoid extraction and HPLC analysis showed that the content ofβ-carotene in CPTA-0.2g/L-24h treated group was as high as1206ug/g DW,while theβ-carotene content in corresponding CK-24h group was only368ug/g DW;.The content of lycopene in CK group was very low,but it was higher in CPTA-0.2g/L-24h group.4.Analysis of the expression level of key genes in carotenoid Metabolic PathwayIn several genes upstream of lycopene synthesis,the expression of Of PSY and Of ZDS in CPTA-0.2g/L-12h group was higher than that in CK-12h group,and that in CK-36h group was higher than that in CK-36h group.The expression of Of ZDS and Of CRISO genes was higher and significantly different than that of CPTA-0.2g/L-36h group,but there was no significant difference between the two groups in other time periods.In several genes downstream of lycopene cyclization,CPTA-0.2g/L-12h contrasted with CK-12h group,The expression of Of LCYE,Of LCYB1,Of CHYE,Of CHYB,Of ZEP and Of NCED1 increased,and the expression of Of LCYE,Of LCYB1,Of CHYE and Of NCED1 increased significantly.At 24 h,the expression of Of LCYE,Of LCYB2,Of CHYE,Of CHYB,Of ZEP in CPTA-0.2g/L-24h was higher than that in CK-24h,and the expression of Of LCYE,Of LCYB2,Of ZEP was significantly higher than that in CK-24h.At h,except Of NCED1,the gene expression in CK-36h group was higher than that in CPTA-0.2g/L-36h group,and the difference was significant.5.Data analysis of transcriptome.According to the data of transcriptome,the key genes of carotenoid metabolism were screened to obtain the transcripts ID and sequences of potential key genes.The results of heat map and correlation between the selected key genes combined with q RT-PCR and carotenoid content were analyzed respectively.it was found that there was no significant difference between CK and CPTA-0.2g/L groups in each time period.On the other hand,the correlation analysis between the transcriptional group data and carotenoid content showed that there was a certain correlation between the selected key genes and the six carotenoids detected,but the change of carotene content in each species.The correlation between chemical and different genes is not consistent.