温州蜜柑完熟栽培期间果实糖酸变化及其相关基因表达分析

Analysis on Changes in Sugar and Organic Acid and Related Gene Expression in Fruit of Satsuma Mandarin during Delayed Harvest Cultivation

【目的】明确温州蜜柑在设施完熟栽培过程中果实糖酸含量的变化规律及其与相关基因表达的关系,揭示其果实品质提升的分子机理。【方法】以温州蜜柑‘宫川’品种为材料,测定完熟栽培期间不同时间果实糖酸含量,并通过荧光定量PCR技术分析果肉中糖及柠檬酸代谢相关基因相对表达量变化,最后对糖酸含量变化与相关代谢基因表达量进行相关性分析。【结果】温州蜜柑果实在设施完熟栽培期间可滴定酸含量不断下降,而可溶性糖含量在花后260 d之前持续增加,之后缓慢下降。高效液相色谱(HPLC)法分析表明蔗糖在果肉所有糖中含量最高,其次分别为果糖和葡萄糖;有机酸则以柠檬酸为主,其含量变化决定总有机酸的含量。糖合成相关基因CitSPSs和CitSUSs相对表达量总体上呈上升趋势,其中CitSUS3及CitSUS4与总糖含量分别呈显著正相关,而CitSPS1、CitSPS2、CitSUS1以及CitSUS6基因表达量与总糖含量变化虽然呈正相关关系但不显著;柠檬酸合成基因CitPEPC和CitCS整体上也呈上升趋势,而相关性分析表明其表达量与柠檬酸含量呈不显著负相关关系;柠檬酸降解基因CitACO3和CitGAD4表达量在完熟栽培过程中均呈上升趋势,且与柠檬酸含量呈显著负相关关系,而CitGS2表达量变化波动较大,与柠檬酸含量相关关系弱。【结论】温州蜜柑果实在设施完熟栽培期间总糖含量不断提高,有机酸含量不断下降,蔗糖含量决定总糖含量,而有机酸含量则由柠檬酸决定;在完熟栽培期间果肉中糖的积累主要由CitSUS3和CitSUS4基因的表达决定,而柠檬酸含量的下降主要由降解基因CitACO3和CitGAD4的上调表达来决定,而与合成基因的表达无直接关联,因此认为完熟栽培期间柠檬酸的降解可能主要通过GABA途径进行。该研究明确了设施完熟栽培的温州蜜柑果实中糖酸积累的变化规律及与相关基因表达的关系,为揭示完熟栽培温州蜜柑果实品质提升的分子机制提供一定的理论基础。

[Objective]This study aimed to determine the changes of sugar and acid content and its relationship with the expression of related genes in the fruit of satsuma mandarin(Citrus unshiu Marc.)fruit during facility delayed harvest cultivation,revealing the molecular mechanism of citrus fruit quality improvement.Citrus facility delayed harvest is a high-efficiency cultivation technique to improve fruit quality,it was valuable to explore the quality change and regulation mechanism.[Method]The fruits of the‘Miyagawa’Satsuma mandarin with uniform size,no pests,mature consistency,and surface without mechanical damage were collected at 220,230,240,250,260,270,and 280 days post-anthesis(DPA)respectively from a commercial orchard in Xin’gan county,Jiangxi province.Soluble sugar was measured by anthrone-sulphuric acid colorimetry,and titratable acid was determined by acid-base neutralization titration.The content of sugar and acid in the fruit were detected with high performance liquid chromatography(HPLC).The expression of genes involved in sugar and citric acid metabolism were analyzed by real-time fluorescence quantitative PCR(qPCR).And finally analyze the correlation between the change of sugar and acid content and the expression of related metabolic genes.[Results]The soluble sugar concentration of fruit increased from 220 DPA to 260 DPA,then decreased.The titratable acid content decreased in the whole delayed harvest cultivation stage.High-performance liquid chromatography analysis showed that the concentration of sucrose was the highest in the total sugar,followed by fructose and glucose;Citric acid was the most important organic acid in the fruit,the change of its content determined total organic acid.The results of the gene expression related to the sugar and citric acid metabolism and correlation analysis revealed that the sugar synthesis-related genes CitSPSs and CitSUSs showed a rising trend.The relative expression of CitSUS3 and CitSUS4 showed a significant positive correlation with total sugar content respectively,while CitSPS1,CitSPS2,CitSUS1,and CitSUS6 genes expression showed a not significant positive correlation with changes in total sugar content.Citric acid synthesis related genes CitPEPC and CitCS expression showed a rising trend and had no significant correlation with citric acid content.The relative expression level of citric acid degradation related genes CitACO3 and CitGAD4 showed an upward trend during the whole of delayed harvest cultivation and have a significant negative correlation with the citric acid content.The CitGS2 gene relative expression level changed irregularly and have no correlation with the citric acid accumulation.[Conclusion]The total sugar content of satsuma mandarin fruit increased during the delayed harvest cultivation period,and the organic acid content decreased.Sucrose determines the total sugar content,while the organic acid was the most important citric acid.The relative expression of CitSUS3 and CitSUS4 genes play an important role in determining the accumulation of sugar in the pulp.The decrease of the citric acid content in the fruit was not directly correlated to the gene expression of the citric acid synthesis,it is mainly influenced by the degradation related genes CitACO3 and CitGAD4.The GABA shunt is the main citric acid degradation pathway.This study revealed the change of sugar and acid accumulation and the relationship with the expression of related genes in facility delayed harvest cultivation satsuma mandarin and provided a theoretical basis for revealing the molecular mechanism of the quality improvement for satsuma mandarin fruit.