高温抑制红心猕猴桃果肉花青素积累的分子机制研究

High temperature areas (Wuhan, Anhui) in summer, it was observed that anthocyanin accumulation was significantly inhibited in Actinidia chinensis which seriously affected fruit quality. In this study, two important transcription factors（TFs）, AcMYB75-like and AcMYB123, which regulate anthocyanin synthesis were screened by RNA-Seq analysis ，it was found that the expression level of TFs decreased significantly at high temperature. In order to explore the reasons for the inhibition of the expression of two TFs, we screened the upstream AcHY5 gene by by Y1H and GUS activation assays. AcHY5 could regulate the expression of AcMYB75-like and AcMYB123 by binding promoters. Previous studies have shown that HY5 is degraded by DDB1-DET1-COP1 complex at high temperature in plants. Therefore, it is speculated that DDB1-DET1-COP1 complex can be induced to degrade AcHY5 under high temperature stress condition, and the activity of AcHY5 directly affects the transcriptional activation of AcMYB75-like and AcMYB123, hindering the accumulation of anthocyanin in fruits. On the basis of existing research, this project aims to explore the relationship between anthocyanin synthesis and temperature in the red-centered kiwifruit by using experimental techniques such as Co-IP, EMSA, transient expression and stable genetic transformation, reveal the molecular mechanism of high temperature inhibiting anthocyanin accumulation, and provide theoretical basis for molecular design and breeding of genetic improvement of Actinidia chinensis.

在夏季高温地区（武汉、安徽），红心猕猴桃花青素的积累受到明显抑制，从而降低了果实的品质。本课题选用常温和高温地区生长的红心猕猴桃内果肉进行RNA-Seq分析，发现调控花青素合成的重要转录因子AcMYB75-like和AcMYB123在高温下表达量显著降低。为了探究这两个转录因子表达受抑制的原因，我们通过酵母单杂交和GUS激活实验筛选得到上游AcHY5基因。AcHY5蛋白可激活AcMYB75-like和AcMYB123基因的表达。已有研究表明HY5在高温下受DDB1-DET1-COP1复合体的降解。由此推测在高温下红心猕猴桃的AcHY5蛋白受降解，从而抑制AcMYB75-like和AcMYB123的转录，最终抑制了花青素的积累。本项目拟利用COIP、EMSA、瞬时表达、稳定遗传转化等技术揭示高温抑制花青素积累的分子机理，为猕猴桃遗传改良分子设计育种提供理论依据。

红心猕猴桃因富含花青素，拥有更高的营养价值和经济价值。夏季高温地区，红心猕猴桃花青素的积累受到明显抑制，严重影响果实的品质。我们选取常温和高温地区生长的100 DAA红心猕猴桃内果肉进行RNA-Seq分析，发现调控花青素合成的重要转录因子AcMYBF110和AcMYB123在高温下表达量显著降低,而AcDDB1表达量显著升高。 瞬时注射35S::AcDDB1OE过表达可以有效抑制红心猕猴桃内果肉花青素的合成；通过Western-blot和cell free实验证明AcHY5蛋白可被以AcDDB1为核心E3复合体降解；酵母单杂交和GUS激活实验结果显示AcHY5可激活AcMYBF110和AcMYB123基因的表达。因此得出结论，高温环境促使AcDDB1泛素降解AcHY5蛋白从而抑制调控花青素的MYB表达，最终阻碍了红心猕猴桃果实花青素的积累。该研究为红肉猕猴桃品种选育提供了理论依据。