红肉猕猴桃AcMYB1功能鉴定及其调控机制研究

Red-fleshed kiwifruit has been accepted as the fine fruit due to the appealing red flesh and superior fruit taste. This type of kiwifruit exhibits huge market potential in China and abroad, and become a new growth point for kiwifruit industry in China. Pigmentation of red-fleshed kiwifruit is inhibited by high temperature during cultivation, which destroys the key commodity attribute of this type of kiwifruit cultivars. In this proposed project, deletion mutants of AcMYB1 promoter will be constructed to reveal how the gene can be regulated at the transcriptional level. Function of AcMYB1 would be elucidated by investigating the effect of gene overexpression and suppressed expression on those pathway gene transcription and anthocyanin accumulation under different temperature. In addition, with help of the interaction between AcMYB1 and AcbHLH, we also try to uncover the regulation mechanism by analyzing the transcriptional activation of structural genes promoted by AcMYB1 responding to different temperatures. Functional analysis of AcMYB1 helps to uncover inhibition mechanism of red-fleshed kiwifruit pigmentation by high temperature, and provides a good chance for genetic modification of kiwifruit pigmentation.

红肉猕猴桃因具有红色果肉且风味极佳而成为猕猴桃中的精品，国内外市场潜力巨大，已成为我国猕猴桃产业发展中的新增长点。在产业栽培中，红肉猕猴桃果肉着色受到高温条件的抑制，严重干扰了这类猕猴桃品种关键商品特性的发挥。本研究拟通过研究红肉猕猴桃花青素途径的重要调节基因AcMYB1启动子区的缺失突变体对不同温度的响应探索温度对其自身转录水平上的调控机制；通过分析不同温度下该基因的超量表达及抑制表达对花青素途径相关基因及花青素积累的影响，阐明AcMYB1的生物学功能；研究AcMYB1与bHLH的相互作用,分析不同温度下AcMYB1对结构基因启动子转录激活能力的影响，明确其与花青素合成相关基因及调节因子的调控模式。AcMYB1功能及温度调控机制的研究将为揭示高温抑制红肉猕猴桃着色机理提供重要的证据，也是开展猕猴桃着色品质遗传改良的重要基础。

本项目通过对MYB转录因子进行遗传转化分析，确定该基因为决定猕猴桃红色果肉及着色程度的关键基因。将相应分子标记在不同果肉颜色杂交后代群体中的分离方式进行分析，结果显示这两个位点在红肉猕猴桃杂交后代中不随果肉颜色发生分离。用不同温度处理‘红阳’果实，结果显示花青素合成随着温度升高而降低，而对于花青素降解产物含量，则随着温度升高而增加。转录组分析的结果进一步验证了花青素合成随着温度升高降低的结果，高温下大部分基因表达下调，只有少部分表达上调。同时发现，一部分热激蛋白及其它转录因子与温度处理密切相关。花青素合成结构基因、多酚氧化酶基因及转运相关基因GST 的表达分析结果说明高温能明显抑制花青素的合成及转运。MYB启动子进行功能分析，发现HSP元件对温度响应起着关键作用。AcMYB1与bHLH的超表达载体共同转化‘红阳’猕猴桃，得到的转基因阳性愈伤组织着色比单独用其中一种转录因子基因转得到的深，说明AcMYB1与bHLH相互作用促进花青素的合成。本研究结果将为揭示高温抑制红肉猕猴桃着色机理提供重要的证据，为红肉猕猴桃的分子育种，及优化果肉着色条件并最终实现红肉猕猴桃的高产稳产奠定基础。