查耳酮异构酶在葡萄果实发育过程中的表达及亚细胞定位研究

The Expression and Subcellular Localization of Chalcone Isomerase in Developing Grape Berries

以‘赤霞珠’葡萄果实为材料,利用制备好的葡萄查耳酮异构酶(CHI;EC 5.5.1.6)CHI多克隆抗体,采用RT-PCR、蛋白质印记杂交和胶体金免疫电镜技术对葡萄果实发育过程中CHI基因、蛋白表达及亚细胞定位进行了研究。结果表明:CHI基因和蛋白的表达随着果实发育有着明显的变化,在果实发育早期和转色期表达量较高,同果实中总类黄酮含量的积累变化一致;胶体金免疫定位显示在果实发育早期CHI主要分布于葡萄果皮细胞的细胞质中,在液泡及质体中也有少量分布;在转色期主要定位于细胞质、质体和细胞核中,其数量明显增多。在成熟果实中,主要存在于细胞质,少量存在于质体和液泡中。

The gene expression, protein accumulation and subcellular localization of Chalcone isomerase （CHI, EC 5.5.1.6） in developing grape berry （ Vitis vinifera L. ＇Cabernet Sauvignon＇ ） was performed via RT-PCR, Western-blotting and immunogold electron microscopy technique using grape CHI antibody. The results showed that the quantity of CHI gene transcript and protein in developing grape berry was developmental stage dependant. The high content of CHI transcripts and protein was observed in grape berries at early development stage （20 days after flower） and veraison, corresponding well to the accumulation of total flavonoids. Immunogold localization result showed that CHI was mainly localized in cytoplasm of the young grape berry skin cells, while some gold particles representing CHI were also found in the vacuole and plastid. In the skin cells of grape berry at veraison, CHI was mainly distributed in cytoplasm, plastids and nuclear, and the gold particles representing CHI increased significantly at this stage; CHI were observed in cytoplasm at late developmental stage, a little was also investigated in the vacuole and plastids.