Cloning And Expression Analysis Of The Key Enzymes Genes Related To Aroma Terpenoids Pathway From Cestrum Nocturnum

Cestrum nocturnum is a fragrant plant belonging to cestrum in solanaceae.Cestrum nocturnum has two specific features with strong aroma and unique behaviors which many physiological activities such as aroma metabolism,circadian rhythm and aroma emission,etc.are contrary to ordinary plants,and becomes an ideal material for exploring mechanism of metabolism of floral aroma and regulation of circadian rhythm.Terpenoids are one of the most important components of specific aroma,their content is only less than aromatic volatile substances whose content is the highest in total floral aromas of Cestrum nocturnum.Biosynthesis of terpenoids in plants is closely associated with the two pathways of MVA and MEP,some key rate-limiting enzymes such as DXS,DXR,GGPPS,HMGR and IPI,etc.located in upstream of the above pathways regulate biosynthesis of terpene precursors and become target enzymes in genetic engineering of plant aroma.The components of floral aroma and their changing trends at different time of opening at night in flowers of Cestrum nocturnum were detected by HS-SPME/GC-MS technology.Full-length cDNAs or cDNA fragments of classic reference genes were cloned by RT-PCR and RACE from and screened as reference genes for subsequent qRT-PCR detection at different stages of flowers and leaves during blooming in 24h-day and night.Full-length cDNAs of genes encoding the rate-limiting enzymes regulating metabolic pathways of terpenoids were isolated by combination of RT-PCR and RACE from Cestrum nocturnum,Dynamic changing of relative expression levels of the isolated genes were detected by qRT-PCR at different stages of flowers and leaves during blooming in 24h-day and night.Plant over-expression vectors were constructed using ORF sequences of the above isolated genes for transformation to tobacco K326 by agrobacterium-mediated leaf disc method to obtain transgenic plants and to identify preliminarily the functions of the transferred genes in the metabolic pathways of terpenoids.This The work could explain the change rules and the molecular mechanism of biosynthesis of terpenoids during aroma emission from the level of transcription and biochemistry,and lay the foundation for the genetic engineering of flower aroma,had important theoretical significance and practical value.Main contributions of this work are as follows:(1)The components and change rules of the floral aroma of Cestrum nocturnum were clarified.the aroma components and the peak area changes was detected by HS-SPME/GC-MS technique using flowers opened at night from Cestrum nocturnum during blooming stage.The results showed that the total peak area of 24:00 was the highest,and increased before 24:00,decreased.after 24:00.Through the analysis we got 22 kinds of compounds with higher matching degree,of which 12 were aromatic compounds,the relative peak area up to 87.48%,benzaldehyde,benzene acetaldehyde,acetic acid benzyl ester as the main component;7 were terpenoids compounds,the relative peak area up to 7.1%,αfarnesene,ocimene,Linalool as the main composition;3 were fatty acid derivatives.(2)The optimal reference genes for the qRT-PCR analysis was cloned,screened and determined from Cestrum nocturnum during flowering.Full-length cDNAs or cDNA fragments of classic reference genes(Actb7,EF-1A,GAPDH,TUA,TUB2,UBQ)were cloned by combination of RT-PCR and RACE from Cestrum nocturnum during blooming stage.The expression of 18S rRNA and the 6 reference genes in flowers and leaves in 24h was analyzed by qRT-PCR method,the stability of this reference genes were analyzed by Bestkeeper,geNorm,NormFinder.The results showed that the expression of Actb7 and EF-1A had the most stability in flowers;EF-1A and UBQ was stable in leaves;EF-1A was stable in all two tissues.among the three groups of analysis,geNorm program showed the optimal number of reference genes was 2 and the best combination was Actb7 and EF-1A.This work used EF-1A as reference gene.(3)Full-length cDNAs of genes encoding the rate-limiting enzymes regulating metabolic pathways of terpenoids was isolated and the relative expression change rules in 24h was analysed by qRT-PCR.The Full-length cDNA sequence of 6 genes(CnDXR,CnDXS1,CnDXS2,CnGGPPSl、CnHMGR1,CnIPI1)encoding the rate-limiting enzymes regulating metabolic pathways of terpenoids was cloned by RT-PCR and RACE technology from Cestrum nocturnum.Sequence analysis showed that the Full-length of CnDXR gene was 1663bp and ORF was 1410bp,encoding 469 amino acids,the full-length of CnDXS1 gene was 2510bp and ORF length of 2136bp,encoding 711 amino acids,the full-length of CnDXS2 gene was 2370bp and ORF was 2145bp,encoding 714 amino acids,the full-length of CnGGPPS1 gene was 1273bp and ORF was 1071 bp,encoding 356 amino acids,the full-length of CnHMGR1 gene was 23 81 bp and ORF was 1821 bp,encoding 606 amino acids,the full-length of CnIPI1 gene was 1108bp and ORF was 855bp,encoding 284 amino acids.The relative expression of these genes in flowers and leaves in 24h was analyzed by qRT-PCR method using EF-1A as reference gene.The results showed that,in the flower,except for CnGGPPS1,5 genes have some rhythmic,among which,CnDXR,CnDXS1,CnDXS2 were more obvious.After 18:00,except for CnGGPPS1,the relative expression of the other 5 genes showed an upward trend,while decreased before 6:00.In leaves,the expression of CnDXS2,CnHMGR1,CnIPI1 has some rhythmic,CnDXR,CnGGPPS1 relative expression of small changes,the relative expression of CnDXS1,CnDXS2 in the night is higher than the day,CnHMGR1,CnIPI1 on the contrary.(4)The transgenic K326 plants was obtained and the function of transferred genes in the metabolic pathways of terpenoids had been identified preliminarily.over-expression vectors were constructed using ORF sequences of the above isolated genes for transformation to tobacco K326 by agrobacterium-mediated leaf disc method and obtained transgenic plants.After Kan screening and DNA,RNA detection by PCR,we got 14 positive T0 plants carrying CnDXR,the positive rate was 41.1%,8 positive T0 plants carrying CnDXS1,the positive rate was 80%,24 positive T0 plants carrying CnGGPPS1,the positive rate was 60%,18 positive T0 plants carrying CnIPI1,the positive rate was 45%.Except for the CnDXS1 plnats showed leaf Chlorosis in seedlings stage and lower in chlorophyll a and carotenoid synthesis,The growth status of the over-expressed tobacco was better than that of ck,and chlorophyll a and carotenoid were higher than ck.