The Study Of Carotenoid Biosythetic Pathway From Citrus Maxima

Citrus maxima Burm.Merr.cv.'Guanximiyou,originated from Pinghe county,Zhangzhou city,Fujian province,which is a famous brand of Citrus maxima in our country.Citrus maxima Burm.Merr.cv.'Hongroumiyou',Citrus maxima Burm.Merr.cv.'Hongmianmiyou' and'Citrus maxima Burm.Merr.cv.Sanhongmiyou,are all the varieties of bud mutation from 'Guanximiyou'.'Guanximiyou','Hongmianmiyou','Sanhongmiyou'and 'Hongroumiyou' were used as the experimental materials in this study,in order to insight into the accumulation of majorcarotenoids in different tissues of Citrus maxima and the relationship between the accumulation and the genes in the synthesis pathway of carotenoids,finally to lay the theoretical foundation for revealing the coloring mechanism of Citrus maxima.The main results were as follows:1.The establishment of carotenoid UPLC detection system for Citrus maxima.Using ACQUITY UPLC(?)(WATERS,company in the United States)ultra high performance liquid chromatography-mass spectrometry instrument with the BEH C18 column,2.1×100 mm,1.7 ?m.Doing isocratic elution with mobile phase contained acetonitrile:methanol:tetrahydrofuran=90:5:5.The detection wavelength:450 nm,flow rate:0.3 mL/min,detection column temperature:40?,sample size:2 ?L and detection time:8 min.This method could be used to separate the carotenoids from different tissues of Citrus maxima,which contributed to its qualilative and quantitative analysis.2.Separation and identification of major carotenoids in Citrus maxima.The main components of carotenoids in the 4 kinds of Citrus maxima were lycopene and ?-carotene,but the accumulated tissue is not exactly same.Lycopene and ?-carotene were mainly accumulated in the segment membrane of 'Guanximiyou'.And in 'Hongmianmiyou',they were mainly accumulated in the segment membrane and albedo.In Citrus maxima cv.'Sanhongmiyou',lycopene was maily accumulated in the juice sacs,segment membrane and albedo,but ?-carotene was maily accumulated in the juice sacs and segment membrane.In'Hongroumiyou',lycopene was maily accumulated in the juice sacs,but?-carotene was maily accumulated in the juice sacs and segment membrane.3.Cloning and bioinformatic analysis of related genes.The ORF sequences of CmPSY,CmPDS,CmZDS,CmCRTISO,CmLCYE,CmLCYB,CmBCH and CmZEP were chose from the data of transcriptome(unpublished),and the sequences were cloned to make up for the lack of basic groups.Bioinformatics analysis showed that the amino acids of the sequences were highly homologous to the same amino acids of Citrus sinensis.4.Using quantitative real-time PCR to analyze the expression of related genes.When lycopene was accumulated largely,the expression quantity of CmPSY,CmPDS,CmZDS and CmCRTISO would improve obviously,and the expression trend of the 4 genes was same.When?-carotene was accumulated largely,the expression quantity of CmLCYE would improve obviously.Generally,when the more lycopene accumulated,the higher expression of CmPSY,CmPDS,CmZDS and CmCRTISO attained.And when the more p-carotene accumulated,the higher expression of CmLCYE attained.