Identification Of Genes Involved In Sugar Accumulation And Component Transformation And Their Regulatory Mechanisms During Pummelo Juice Sacs (Citrus Grandis 'Seedless') Development

Citrus grandis 'seedless' now is widely cultivated in Hainan province,China.The essential features of this cultivar are thin skin,green epicarp and seedless at the ripening stage.Here,using C.grandis "seedless" as experimental material,we investigated the physical and inclusion indicators,and found that the rate of fruit weight increase was faster than that of fruit volume increase due to the accumulation of compounds in 120?210DAF.Component analysis of soluble sugar by HPLC indicated that arabinose have a high content in early development of pummelo juice sacs(PJS),whereas fructose,glucose and sucrose show a significant increase during PJS maturation.To clarify a global overview of the gene expressing profiles,the PJSs from four periods(60,120,180 and 240 DAF)were selected for comparative transcriptome analysis.The resulting 8275 unigenes showed differential expression during PJS development.Based on transcriptome data,resulting in a set of 5 genes(UBC,ACT,OR23,DWA2 and CYP21D)could be used as internal reference genes for qRT-PCR analysis of PJS.Sucrose is the end product of photosynthesis.Upon translocation through the phloem to sinks,sucrose is degraded by either sucrose synthase(SUS)or invertase(INV).To study the function of SUS and INV genes in PJS,we used the C.grandis'seedless' as material,Based on Arabidopsis SUS and INV proteins,we used BLASTX to search the homologous protein in pomelo genome.A total of 6 Cg-SUS and 12 Cg-INV with complete open reading frame(ORF)were identified.Based on their positions on C.grandis chromosomes,genes were named as Cg-SUS1?6 and Cg-INV1?12,respectively.Additionally,the analysis on its physicochemical properties,intron and exon,phylogenetic tree,and amino acid functional domain was performed.The digital expression analysis indicated that Cg-SUS2,Cg-SUS3 and Cg-INV6 showed an active transcript in juice sacs during fruit development of C.grandis 'seedless',and also their expression profiles were detected by qRT-PCR.Combined with the correlation analysis between the sugar content and the gene expression,the results showed that the expression of Cg-SUS2 has established a close positive correlation between the content of xylose and arabinose.We believe Cg-SUS2 might be involved in biosynthesis of arabinose and xylose.The expression of Cg-SUS3 and Cg-INV6 has established a close positive correlation between the content of glucose,fructose and sucrose,suggesting that Cg-SUS3 may be involved in the degradation of sucrose,which is converted into UDP-glucose and fructose.And Cg-INV6 may be a key enzyme in vacuolar hexose accumulation.LEC1 is a member of NF-YB transcription factor.In this study,LEC1 protein sequences from Arabidopsis thaliana database were compared by BlastX with C.grandis 'seedless' transcriptome data,and 5 of LEC1 homologous sequences were screened out for Gene expression profile analysis.So as to design full-length CgNF-YB9 primers,conduct full-length cloning and construction of eukaryotic expression vectors(pCgNF-YB9),and use transient expression Nicotiana tabacum L.leaves and qRT-PCR technology to detect the influence of related gene expression on the process of sugar metabolism.The qRT-PCR results showed that overexpression of CgNF-YB9 may affect the expression of genes related to sucrose synthesis and sucrose degradation,which laid a foundation for future transgenic studies on C.grandis 'seedless'.