枣果实成熟过程中类黄酮的积累及相关基因的表达分析

The patterns of flavonoids accumulation and the expression of biosynthesis related genes during the course of maturation of the Chinese jujube fruit

【目的】探究枣果实成熟过程中类黄酮的积累及相关基因的表达模式,为枣功能成分类黄酮的开发利用提供依据。【方法】以‘稷山板枣’和‘骏枣’为材料,采用分光光度法测定两个品种果实成熟过程中果皮和果肉的总黄酮和总黄烷醇含量,利用HPLC检测类黄酮组分的含量变化,qRT-PCR分析类黄酮合成相关基因的表达。【结果】‘稷山板枣’和‘骏枣’成熟过程中,果皮和果肉的总黄酮和总黄烷醇含量逐渐下降,干枣时期有所增加,果皮中含量高于果肉,‘稷山板枣’含量高于‘骏枣’;两个品种果皮和果肉中类黄酮组分基本相同,黄烷醇和黄酮醇含量从白熟期逐渐下降,部分黄酮醇组分含量在成熟后期(半红/全红)含量有所上升;类黄酮合成相关基因在两种枣果皮中的表达量从白熟期到完熟期呈下调趋势,但品种间及不同成熟时期的表达有所差异。相关性分析结果显示,下游基因与类黄酮及其组分含量呈显著或极显著相关。【结论】两个枣品种成熟过程中,类黄酮积累模式基本一致,枣果皮较果肉含量高,且均在果实成熟早期积累最高,随果实成熟而下降;结构基因F3H、F3’H可能对类黄酮的合成积累具有关键促进作用,LAR可能是黄烷醇途径中的关键调控基因。

【Objective】Chinese jujube(Ziziphus jujuba Mill.)is an economic forest fruit tree in the Rhamnaceae.The jujube production in China accounts for more than 90%in the world.Jujube fruits are rich in flavonoids.The study aimed to explore the accumulation pattern of the flavonoids and the expression of related genes during the ripening of jujube fruit,and provide a basis for the development and utilization of functional components in jujube fruits.【Methods】The experimental materials’Jishanbanzao’and’Junzao’were obtained from the Jujube Experimental Station of Northwest A&F University in Qingjian,Shaanxi,China.The jujube fruits at different stages(white maturity,beginning-red,half-red,red maturity,full maturity,dry date period)were separated into peel and pulp.The samples were immediately frozen in liquid nitrogen and stored in the ultra low temperature refrigerator at-80℃.The contents of total flavonoids and total flavanols in the peel and the pulp of two jujube varieties at different degree of maturation were determined by spectrophotometry,and the change of each flavonoid component content was detected by HPLC.qRT-PCR was used to analyze the expression of flavonoid synthesis-related genes.【Results】During the whole maturation process of’Jishanbanzao’and’Junzao’,the content of total flavonoids in the peel and the pulp showed a continuous downward trend as the fruit matured,and it increased slightly in the dry date period.The content of total flavonoids of’Jishanbanzao’was higher than that of’Junzao’in all stages.The total flavonoids content in the pulp of the two varieties was much lower than that of the peel(the difference was 3.27 to 36.90 times).The total flavanols content in the peel of the two varieties was the highest in the white maturity period,and decreased slowly at the beginning red stage,and reduced greatly in the half-red stage and full red stage.The loweast content was found in the dry date period,only about 1.5%of the initial ripening content.During the ripening process,the total flavanol content in the pulp was always lower than that of the peel but it increased in the dry date period which was higher than that in the peel.Four kinds of flavanols and five kinds of flavonols were detected in the peel and pulp of’Jishanbanzao’and’Junzao’by HPLC.Flavanols were catechin,epicatechin,procyanidin B1 and procyanidin B2.The contents of catechin in the two varieties were the highest in the peels and pulps,the content of proanthocyanidin B2 in the peel was the lowest,and the contents of epicatechin and proanthocyanidin B2 in the pulp were lower.The four flavanol substances in the peels gradually decreased with the ripeness of the fruits and the contents decreased to the minimum value at the stage of dry date.The change trends of the four flavanol components in the pulps from white maturity stage to the full maturity stage were consistent with the peels,showing a continuous decline,but it increased significantly in the dry date period.Quercetin and its glycoside derivatives were the main flavonols.Quercetin and its glycosides showed a downward trend as a whole from the white maturity stage to the full maturity stage in the peels of the two jujube varieties,but the contents of some substances increased slightly when they were half-red or full-red.The contents of all flavonol components in the pulps were significantly lower than that in the peel,and gradually decreased from the white maturity stage,but the content increased in the dry date period.qRT-PCR analysis found that the changes in the expression of flavonoid synthesis-related genes in the peels of the two jujubes were basically the same.There was a downward trend from the white maturity to the full maturity stage,but the expression varied with the varieties and the different maturity periods.The correlation analysis results showed that the downstream genes(F3 H,F3’H,FLS,ANR,LAR,UFGT)were significantly or extremely significantly related to the content of flavonoids and the components.【Conclusion】The accumulation patterns of flavonoids in the two jujube varieties during maturation were basically the same with slight differences.The contents of the total flavonoids in the peel were higher than that in the pulp(the difference was about 20 times).The accumulation of flavonoids was the highest in the early stage of fruit ripening,and it decreased with the ripening of the fruit.Structural genes showed a downward trend as the fruit ripening,and downstream genes were significantly correlated with the flavonoid content.The structural genes F3 H and F3’H might play a key role in promoting the synthesis and accumulation of flavonoids,and LAR might be the key regulatory gene in the flavanol pathway.