生长素输出载体蛋白PIN1在作物根和胚中的亚细胞定位

Subcellular localization of auxin efflux carrier protein PIN1 in crop root and embryo

关键词:  生长素输出载体, PIN1, 水稻, 小麦, 玉米, 大豆

Key words:  auxin efflux carrier, PIN1, rice, wheat, maize, soybean

摘要:

生长素输出载体在植物发育中起非常重要的作用。然而,生长素输出载体蛋白PIN1在农作物水稻、小麦、玉米和大豆的根和胚中的亚细胞定位尚不清楚。该研究首先分析了OsPIN1b和它的同源物的氨基酸序列特征,发现小麦(TaPIN1)、玉米(ZmPIN1b)和大豆(GmPIN1b)中的PIN1序列与水稻的OsPIN1b序列分别具有61.5%、62.5%、61.9%的相似性。然后根据水稻‘日本晴'(‘Nipponbare')的OsPIN1b的氨基酸序列,人工合成OsPIN1b多肽并注射健康的新西兰白兔获得了抗兔的OsPIN1b多克隆抗体,在通过免疫印迹方法检测抗兔的OsPIN1b多克隆抗体的有效性后,发现可以利用该抗体有效检测到水稻叶片及根中OsPIN1b的表达。为检测OsPIN1及其同源物在不同作物胚根和胚中子叶细胞的定位,利用制备的抗兔的OsPIN1b多克隆抗体并通过免疫组化实验,发现水稻的OsPIN1b、小麦的TaPIN1和玉米的ZmPIN1b非极性定位在早期的胚根和胚中子叶表皮细胞的细胞质膜上,大豆中的GmPIN1b非极性定位在胚根表皮细胞的质膜上,而在胚的子叶细胞中是胞质定位。为进一步检测水稻中OsPIN1b的亚细胞定位,对水稻根分生区表皮细胞用蛋白质转运抑制剂BFA(Brefeldin A)及抗兔的OsPIN1b多克隆抗体处理后,进行免疫组化实验,结果发现水稻中的OsPIN1b可以通过胞吞转运途径从水稻根表皮细胞膜进入细胞质中。该研究利用抗兔的OsPIN1b多克隆抗体有效检测了OsPIN1b及其同源物在水稻、小麦、玉米和大豆的胚根表皮细胞及胚中子叶表皮细胞的亚细胞定位,这将有助于进一步揭示生长素输出载体OsPIN1b及其同源物通过调控生长素极性运输而参与作物发育的作用机制。

Abstract:

Auxin efflux carrier plays an extremely important role in plant development. However, the subcellular localization of auxin efflux carrier PIN1 in the roots and embryos of crops rice, wheat, maize and soybean remains unclear. In this study, the characterization of OsPIN1b and its homologous amino acid sequence were analyzed, and it showed that the PIN1 sequences of wheat(TaPIN1), maize(ZmPIN1b)and soybean(GmPIN1b)shared 61.5%, 62.5% and 61.9% similarities with rice OsPIN1b, respectively. Next, an artificial OsPIN1b polypeptide was synthesized based on the OsPIN1b amino acid sequence of rice ‘Nipponbare' and injected it into healthy New Zealand white rabbits to obtain anti-rabbit OsPIN1b polyclonal antibody. The effectiveness of the prepared polyclonal antibody against OsPIN1b was detected by immune blot method, and the expression of OsPIN1b was found to be effectively detected in rice leaves and roots. Furthermore, the subcellular localization of OsPIN1b and its homologous in primary roots and cotyledon cells of embryos in different crops was detected with anti-rabbit OsPIN1b polyclonal antibody by immunohistochemistry assay. The results showed that rice OsPIN1b, wheat TaPIN1 and maize ZmPIN1b apolarly localized on the plasma membrane of epidermal cells of primary roots and cotyledon of embryo in rice, wheat and maize grown in early development stages, and soybean GmPIN1b apolarly localized on the plasma membrane of primary root epidermal cells, but was cytosolic localization in the cotyledon cells of embryo. To further detect the subcellular localization of OsPIN1b, epidermal cells of rice primary root meristem region were treated with protein transport inhibitors BFA(Brefeldin A)and anti-rabbit OsPIN1b polyclonal antibody and detected by immunohistochemistry assay. It showed that OsPIN1b localized on cytoplasma membrane of rice root epidermal cells could enter into the cytoplasm via endocytic trafficking manner. In this study, the subcellular localization of OsPIN1b and its homologous in the epidermal cells of primary roots and cotyledons of embryos of rice, wheat, maize and soybean were effectively detected with the anti-rabbit OsPIN1b polyclonal antibody, and it will facilitate us to reveal the molecular mechanism of auxin efflux carrier OsPIN1b and its homologous by regulating polar auxin transport to involve in crops development.