Cloning Of Mango Genes Encoding Genarnylgeranyl Pyrophosphate Synthases And Preliminary Studies On Their Relationships With Carotenoids Biosynthesis

Geranylgeranyl diphosphate(GGPP)is the precursor for the biosynthesis of carotenoids,chlorophylls and other isoprenoid in plants.The isoprenoid is mainly responsible for the quality of fruits and through the excavation of the related genes of the isoprenoid,it provides an important reference for improving flavor and quality of the fruit.In this study,Jinhuang,Guifei,Yexiang,Hongyu and Tainong flesh were used as research materials.Techniques of physiology,biochemistry and molecular biology are used to research regulatory mechanism of GGPPS gene about carotenoids accumulation during ripening of flesh.The results are as follows:1.We cloned the cDNA of a GGPPS homologous gene by using rapid amplification of cDNA ends(RACE)technology,and the theoretical length of cDNA was 1247 bp.The length of open reading frame was 110bp and encoding 369 amino acids.Protein sequence comparison indicated that this GGPPS is closely related(78%)to the GGPPS,(AAM21639.1)of Cistus creticus and the sequence analysis showed that the sequence contained two DDx(2-4)D conserved motifs(FARM and SARM)and a CxxxC motif.The results of cluster analysis showed that this gene was clustered with GGPPS large subunit,so it was speculated that it was an encoding GGPPS large subunit in mango and named it MinGGPPSlsu.Gene structure analysis showed that MinGGPPSlsu had no intron.A subcellular localization plasmid was constructed for transient transformation of tobacco leaves.The gene was localized in chloroplasts by real-time confocal microscopy.We cloned the cDNA of a GGPPS homologous gene by using rapid amplification of cDNA ends(RACE)technology,and the theoretical length of cDNA was 1298 bp.The length of open reading frame was 1002bp and encoding 333 amino acids.Protein sequence comparison indicated that this GGPPS is closely related(83%)to the GGPPSssu(KMl08317)of Azadirachta indica,a species close to Mangifera indica,and the sequence analysis showed that the sequence contained a DDx(2-4)D conserved motif(FARM)and a CxxxC motif.Cluster analysis showed that this gene was clustered with significant independent branch of GGPPS small subunit gene,suggesting that it was encoded as a small subunit of GGPPS in mango and named it MinGGPPSssu.Gene structure analysis showed that MinGGPPSssu had 515 bp intron.A subcellular localization plasmid was constructed for transient transformation of tobacco leaves.The gene was localized in chloroplasts by real-time confocal microscopy.2.Choosing immature and mature fruits of Jinguang,Guigei,Yexiang,Hongyu and Tainong as materials,the results showed that the expression of MinGGPPSlsu and the content of total carotenoids in mature fruit of 5 mango varieties were very higher than immature fruit.The fruit of 6 stages of mature process of Hongyu and Yexiang with different color flesh of mature fruit was used as experimental material.The expression level of MinGGPPSlsu was significantly correlated with the content of total carotenoids,and the expression level of MinGGPPSssu was not related to the content of carotenoids.Indicating that MinGGPPSlsu in mango flesh is involved in the synthesis of carotenoids and its mechanism of action remains to be further studied.3.The colour of Yexiang flesh turns from white to orange,and Hongyu turns from white to yellow during fruit ripening.HPLC analysis of ?-carotene and lycopene:(3-carotene of Hongyu and Yexiang gradually increased and was significantly correlated with MinGGPPSlsu;lycopene was detected in stages 5 and 6 of Yexiang and no lycopene was detected in all stages of Hongyu.This study preliminary explored the effect of GGPPS gene on the accumulation of carotenoids in flesh during ripening.We hope to provide experimental support,and to provide a theoretical basis for the regulation of the production of mango and carotenoids accumulation.