微丝结合蛋白AtVLN1负调节拟南芥耐盐性的作用及其机制

Microfilament (MF) cytoskeleton is an important regulator in almost all cell activities. In 2010, we reported that MF dynamic can directly affect plant salt stress tolerance. Although there are hundreds of actin-binding proteins (ABPs) in planta, olny ARP2/3 is reported on its role in Arabidopsis salt stress tolerance to this day. Thus, it is important to identify new ABPs and understand their role in salt stress. In our earlier studies, the expression of 46 Arabidopsis actin-binding protein genes and the growth of their mutants in salt stress were analyzed. We found that AtVLN1 may play as a key negative regulator of salt response in Arabidopsis. The project will study the expression pattern of AtVLN1 gene and protein in salt stress by qRT-PCR, western blot, reporter gene GFP and GUS methods. In addition, the MF dynamic, the growth and the expression of salt stress-responsive genes in Atvln1 mutants, retrieved mutants, over-expression plants of AtVLN1 gene will be observed in salt stress treatments by MF dynamic observed and other methods. Lastly, the function of AtVLN1 will be analyzed on regulation of ADF (actin depolymerizing factor) family members under salt stress, including their expression, their regulation of the growth and MF dynamic by genetic methods. These results will explain the negative regulation mechanism of actin-binding protein AtVLN1 in Arabidopsis salt stress tolerance, providing the novel data for the molecular mechanism of MF role in plant salt stress tolerance.

微丝-胞内活动的调节子-可能从多途径调控植物耐盐性。2010年申请人首次证实其动态变化直接决定植物耐盐性。至今在几百种微丝结合蛋白(ABPs)中，只证实了拟南芥ARP2/3的抗盐功能，因此亟待鉴定新的盐响应ABPs，并研究其功能。申请人前期从基因表达和突变体表型角度探讨了46种拟南芥ABPs盐响应情况，初步明确AtVLN1是关键的负响应盐因子。 本项目拟在对照和盐处理下，利用qRT-PCR、Western blot、GFP定位和GUS染色方法，研究AtVLN1表达模式；利用活体观察微丝等技术研究Atvln1突变体，恢复突变体，过表达等植株的微丝动态、生长表型和对已知盐响应基因表达量影响；以及利用遗传学手段分析AtVLN1对ADF(微丝解聚因子)家族成员的功能调节，包括表达、生长和微丝动态等。旨在阐明AtVLN1负调节拟南芥耐盐性的作用及其机制，为盐胁迫下微丝作用的分子机理提供科学理论依据。

微丝骨架作为众多细胞内活动的调节因子，可能通过多条途径参与植物适应盐胁迫过程中。2010年本人率先利用药理学实验证明：微丝的动态变化直接影响植物的耐盐性，然而至今盐胁迫下微丝作用的分子机理尚不清楚。AtVLN1是拟南芥5个微丝结合蛋白VLN家族成员之一，体外试验证实其具有捆绑微丝的作用，但是体内的生理功能尚不清楚。.本项目通过荧光实时定量PCR（RT-PCR）、Western Blot，荧光蛋白GFP、GUS染色等方法，发现AtVLN1在各种组织中均有表达，盐胁迫下AtVLN1的表达下降；进一步发现AtVLN1突变提高了植株对盐胁迫的抗性，以及植株的含水量，推测在盐胁迫下AtVLN1可能发挥负向调节作用，并且通过影响植株耐受盐胁迫过程中的渗透调节作用，从而实现功能。通过研究AtVLN1对植株各组织器官细胞形态观察，结果发现AtVLN1通过捆绑微丝作用，在根毛生长中起到负向调节因子，并且在转录水平上受到GL2的直接调控，同时盐胁迫下AtVLN1可能与微丝解聚因子AtADF10相互作用实现对微丝动态的调控作用。因此，本研究结果证实了，盐胁迫下AtVLN1受到GL2的转录调控，表达量下降，同时导致AtADF10表达上升，AtVLN1和AtADF10同时作用导致植物失去了根毛生长过程中捆绑微丝的活性，根毛生长时间变长，从而根毛长度增加，吸收水分含量增加，提高了植物在耐受盐胁迫过程中的渗透胁迫能力，从而提高植物耐盐能力，本研究结果阐明了盐胁迫下AtVLN1的作用及其机制，为AtVLN1的生物学功能，植物适应盐胁迫机理，根毛生长机理提供了重要的科学依据。