关键词: 拟南芥 , 生长素运输 , 内体分选复合物 , 蛋白互作 , 泛素分离系统 , 双分子荧光互补试验

Abstract: 【Objective】The objective of this study is to identify the phenotype of homozygous mutant vps25 under auxin stress conditions, get the interacting protein of Arabidopsis vacuolar protein sorting AtVPS25, and analyze the function and molecular mechanisms of their interaction in the process of auxin response.【Method】The "three primer method" was adopted to identify the mutants. The function of the VPS like protein, AtVPS25, was analyzed by identification of the responses of AtVPS25 mutants to exogenous auxin. AtVPS25 protein was used as a bait to screen its interacting proteins by the split-ubiqutin system in Arabidopsis. The interaction between AtVPS25 and AtAIR12 (for Auxin - Induced Root in cultures) was confirmed by the yeast interaction experiment and the bimolecular fluorescence complementation test (BiFC). The subcellular locations of AtVPS25 and AtAIR12 protein was analyzed by the confocal scanning. The expression patterns of auxin transport-related genes in AtVPS25 mutants were identified by real-time PCR. The subcellular locations of AtVPS25 and AtAIR12 protein were analyzed in plants cells; The expression of part of auxin transport related genes in vps25 mutant under auxin treatment condition were identified by real-time PCR. 【Result】Real-time PCR results showed that under 10 μmol?L-1 IAA treatment conditions, in wild-type Arabidopsis thaliana (WT), the AtVPS25 expression level was increased with the stress time, and reached the highest at 12 h , about 40 times the size of 0 h, which proved that AtVPS25 was induced by auxin treatment. The AtVPS25 protein was used as a bait to screen the interacting protein from Arabidopsis by using the split-ubiqutin system and get the interacting protein AtAIR12. Two-hybrid interaction tests of AtVPS25 and AtAIR12 protein full-length sequence proved that AtVPS25 interacted with AtAIR12. Subcellular localization test proved that AtVPS25 is located on the cell membrane and cytoplasm, AtAIR12 is located on the cell membrane and chloroplast membrane. BiFC (bimolecular fluorescence complementation) test results showed that AtVPS25 is interacted with AtAIR12, and the interaction sites were on the cell membrane and cytoplasm. Molecular identification showed that homozygous mutant vps25 was obtained. When vps25 growth in conditions of 0.1 mg?L-1 IAA, the elongation of primary root was inhibited, and the difference in primary root was relatively significant (P＜0.01) compared with WT in the same condition, while no significant difference in the number of lateral roots, which have been reported the similar phenotype of the mutant air12-1 under the stress conditions of auxin. When treated with 10 μmol•L-1 IAA, under WT background conditions, the expression level of AtAIR12 responsed obviously for IAA, and increased with the stress time, reached a maximum at 12 h, about 80 times as 0 h, which proved that AtAIR12 and AtVPS25 have the exactly same change trend in WT under conditions of 10 μmol•L-1 IAA treatment. Under the background of vps25 mutant, the expression of AtAIR12 was restrained relative to the WT, the expression level had no obvious change from 0 to 24 h. Additionally, the expression level of auxin efflux carrier gene (AtPIN2) was reduced, while the auxin input vector gene was increased in mutant vps25.【Conclusion】 IAA had the effects of inducing the expression of vacuolar sorting protein gene AtVPS25 in Arabidopsis and AtVPS25 regulated the development of plant primary root. AtVPS25, which interacted with AtAIR12 at cell membrane and cytoplasm, regulated the expression of some IAA associated genes. Results indicated that AtVPS25 had influence on the response of IAA in plant root through regulating expression level of downstream genes above-mentioned. Further research is required to clarify the regulatory mechanism between AtVPS25 and AtAIR12 in plants.

Key words: Arabidopsis thaliana , auxin transport , endosomal sorting complex required for transport , protein interactions , split-ubiqutin system , bimolecular fluorescence complementation experiment test