拟南芥液泡分拣蛋白AtVPS25调控植物生长素响应的功能分析

摘要

【目的】鉴定生长素胁迫条件下纯合突变体vps25的表型，获得拟南芥液泡分选蛋白 AtVPS25的互作蛋白，并分析AtVPS25和其互作蛋白在生长素响应过程中的功能及其分子机制。【方法】根据“三引物法”鉴定突变体；通过观察拟南芥vps25突变体在外加生长素的培养基上的表型，鉴定AtVPS25的功能；以AtVPS25为诱饵蛋白，采用泛素分离系统筛选在拟南芥中与其互作的蛋白；利用酵母互作试验和双分子荧光互补试验（BiFC）验证AtVPS25与AtAIR12（for Auxin-Induced in Root cultures）的互作关系；鉴定AtVPS25和AtAIR12蛋白在植物细胞中的定位情况；采用Real-time PCR方法，分析在生长素处理条件下，部分生长素运输相关基因在拟南芥vps25突变体中的表达变化。【结果】Real-time PCR结果显示，10μmol·L-1 IAA处理条件下，在野生型拟南芥（WT）中，AtVPS25的表达量随着胁迫时间的增长而增高，并在12 h达到最高，约为0 h的40倍，证明AtVPS25受生长素处理的诱导表达。利用泛素分离系统筛库获得AtVPS25的互作蛋白AtAIR12、AtVPS25与AtAIR12全长蛋白序列的酵母双杂交试验证明AtVPS25与AtAIR12互作。亚细胞定位试验证明AtVPS25定位在细胞膜和细胞质中， AtAIR12定位在细胞膜及叶绿体膜上。BiFC（双分子荧光互补）试验结果显示，AtVPS25蛋白与AtAIR12蛋白互作，并且互作位点在细胞膜和细胞质中。突变体鉴定获得纯合突变体vps25。vps25在0.1 mg·L-1IAA条件下生长，表现为主根伸长受到抑制，并且相对同一条件下的WT的主根长度差异极显著（P＜0.01），而同时侧根数无明显差异，这与已报道的air12-1突变体在生长素处理条件下的表型相似。10μmol·L-1 IAA处理时，在WT背景条件下，AtAIR12的表达量对IAA响应明显，并且随着胁迫时间的增长而增高，在12 h时达到最高，约为0 h的80倍，证明10μmol·L-1 IAA处理条件下，WT中AtAIR12表达量的变化趋势与AtVPS25完全相同。同时在vps25突变体背景条件下，AtAIR12的表达相对于WT受到抑制，在0-24 h表达量无明显变化。此外，在 vps25突变体背景条件下，生长素输出载体基因 AtPIN2相对于 WT 中表达量降低，生长素输入载体基因AtLAX2相对于 WT中表达量提高。【结论】拟南芥液泡分拣蛋白基因AtVPS25受 IAA诱导表达，参与调控植物主根的发育，AtVPS25可以与生长素响应蛋白AtAIR12在细胞质和细胞膜上互作，AtVPS25调控部分生长素相关基因的表达，AtVPS25通过调控这些下游基因的表达影响生长素在根部的响应。AtVPS25与AtAIR12的调控机制需要进一步深入研究。%Objective]The objective of this study is to identify the phenotypeofhomozygous mutant vps25 under auxin stress conditions, get the interacting protein ofArabidopsis 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 ofAtVPS25 mutants to exogenous auxin. AtVPS25 protein was used as a bait to screen its interacting proteins by the split-ubiqutin system inArabidopsis. 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 inAtVPS25 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 invps25 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 fromArabidopsisby 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 vps25was obtained. Whenvps25 growth in conditions of0.1 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 levelof 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 geneAtVPS25 inArabidopsis and AtVPS25 regulated the development of plant primary root.AtVPS25, which interacted with AtAIR12at cell membrane and cytoplasm, regulated the expression of some IAA associated genes. Results indicated thatAtVPS25 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.