拟南芥CDPK基因WDSS1参与干旱胁迫反应的实验证据

摘要

干旱是影响植物生长和作物产量的主要因素之一。植物在响应干旱胁迫过程中的一种机制是通过气孔关闭降低水分流失，以保持植物体内的生物活性。在这个过程中，保卫细胞中自由的Ca2+浓度增加，从而使气孔关闭。作为Ca2+的感受器之一，钙依赖的蛋白激酶(CDPK)在这个过程中起着重要的作用。对拟南芥CDPKs参与植物响应水分胁迫的研究还很少。
　　 拟南芥CPK基因突变体wdss1(water deficit stress sensitive1)在水分不足的条件下显示了较野生型更敏感的表型和较高的蒸腾速率。GFP融合蛋白表达亚细胞定位结果表明WDSS1是质膜定位。RT-PCR的结果显示WDSS1是干旱胁迫诱导表达的。Ca2+，H2O2和ABA处理后，wdss1的气孔不能关闭，这与干旱条件下wdss1体内水分大量流失是一致的，说明WDSS1功能的缺失减弱了保卫细胞响应干旱的信号转导过程。酵母双杂交筛选结果证实WDSS1与过氧化氢酶(CATx)在体外互作，但与其它的CAT成员并不互作。BiFC的实验结果也证明了二者在体内是互作的。表型观察结果显示catx与wdss1在干旱条件下都表现出敏感的表型。wdss1体外H2O2的清除率部分降低。过表达株系的H2O2的清除率却较野生型高，表明WDSS1通过激活CATx来增加对ROS胁迫的耐受性。DAB染色结果表明wdss1和CATx突变体在干旱条件下积累较高的H2O2，说明二者参与了调控H2O2平衡的过程。对两种突变体在种子萌发和幼苗生长期间对H2O2和ABA的敏感性实验结果显示差异不明显。过氧化氢酶活性的测定结果显示wdss1与野生型的差异不明显。
　　 以上证据表明WDSS1和CATx在干旱条件下调节气孔运动方面起着重要的作用。体内和体外的实验证据也表明WDSS1与CATx是互作的，推测WDSS1可能通过磷酸化CATx，并控制H2O2的平衡。

目录

文摘

英文文摘

论文说明：Abbreviations

声明

Chapter 1: Research Background

1.1 Stomatal Regulation

1.1.1 Stomatal Opening

1.1.2 Stomatal Closing

1.1.3 ABA signaling in guard cells

1.1.4 H2O2 as a signal molecule in guard cells

1.1.5 Calcium: A central regulator in signal transduction cascades

1.2 Calcium signaling through protein kinases

1.2.1 Calcium dependent protein kinases (CDPKs)

1.2.2 CDPK-related kinases (CRK)

1.2.3 Calmodulin-dependent protein kinases (CaMKs) and calcium and calmodulin-dependent protein kinases (CCaMK)

1.3 Objectives of the study

Chapter 2: Guard cell expressed CDPKs and their response to drought

2.1 Introduction

2.2 Materials

2.2.1 Chemicals

2.2.2 Plant materials

2.3 Methods:

2.3.1 Preparation of plant materials

2.3.2 Measurement of water loss

2.3.3 Stomatal Aperture Measurements

2.3.4 Drought Measurements

2.4 Results:

2.4.1 Measurement of water loss from excised Leaves

2.4.2 Stomatal Aperture Measurements

2.4.3 Phenotype observation after drought treatments

2.5 Discussion

2.5.1 Water loss from excised Leaves

2.5.2 Stomatal Regulation

2.5.3 Drought Sensitivity

Chapter 3:WDSS1 involved in stomatal regulation under water stress

3.1 Introduction

3.2Materials and Methods

3.2.1 DNA Extraction from leaf materials

3.2.2 RNA Extraction

3.2.3 Overexpression, Complement, WDSS1-GFP, and CATX-GFP

3.2.4 Drought tolerance measurements

3.2.5 Water loss Measurements

3.2.6 Stomatal aperture measurements

3.2.7 Measurement of H2O2 Production

3.2.8 Measurement of H2O2 elimination by whole plant

3.2.9 Sensitivity to H2O2 and ABA during germination and seedling development

3.2.10 Yeast two-hybrid assay

3.2.11. Constructions for Yeast Two Hybrid 2nd Screening

3.2.12. Measurement of Catalase Activity

3.3 Results

3.3.1 Drought Sensitivity

3.3.2 Water loss measurements of detached leaves

3.3.3 Stomatal aperture measurements

3.3.4 Sensitivity to ABA during germination and seedling development

3.3.5 Sensitivity to H2O2 during germination and seedling development

3.3.6 Yeast Two Hybrid Assay to find down stream target

3.3.7 Subcellular Localization of WDSS1 and CATX

3.3.8 WDSS1 Mutants Produce More H2O2

3.3.9 Measurement of H2O2 elimination by whole plant

3.3.10 Catalase activity of wild-type and wdssl plants

3.3.11 catx mutant allele showed phenotype similar to wdssl mutants

3.4 Discussion

References

ACKNOWLEDGEMENT