钙依赖型蛋白激酶在杨树耐旱和耐盐中的调节作用

The phyto-hormone abscisic acid (ABA) plays a central role in plant adaptation to abiotic stress such as drought and salinity. Previous reports had shown that the calcium-dependent protein kinases (CDPKs) have an important role in ABA signal transduction that is important for the plant adaptation to drought and salt tolerance..Populus has been widely used model as it was the first forest tree genome to be sequenced. Furthermore, Populus has a rapid growth, extensive root system, high perennial biomass production and enormous economical importance. In this project we will use Populus x canescens, Populus popularis and Populus euphratica as experimental model in order to study the regulation mechanism of CDPKs in Poplar adaptation to drought and salt tolerance..The first objective will be focused in identifying the candidate CDPKs genes by screening which of them response to ABA, drought and salt treatment. Aditionally, we will determine their tissue and subcellular localization. Later on, we will use transgenic technology to obtain the RNAi and overexpression lines of the candidate CDPKs in Populus x canescens, we will also use the orthologous gene expresion in Arabidopsis to assay the ABA-related phenotypes and to identify the function of those candidate CDPKs. Lastly, to study the molecular mechanism of candidate CDPKs in poplar adaptation to drought and salt stress we will screen the putative interaction proteins of CDPKs in yeast two hybrid system. The interaction will be confirmed by co-immunoprecipitation, bimolecular fluorescence complementation and tobacco luciferase complementation. Finally we will use of in vitro phosphorylation to confirm that CDPKs interacting proteins are also substrates.

脱落酸（abscisic acid, ABA）在植物适应干旱胁迫和盐胁迫过程中具有重要作用，钙依赖型蛋白激酶（calcium-dependent protein kinase, CDPK）是ABA信号转导过程中重要的调控因子。.本项目以灰杨、群众杨和胡杨为实验材料，研究CDPK在杨树耐旱和耐盐中的调节作用。首先，筛选响应ABA、干旱和盐处理的CDPK作为候选基因，确定杨树候选CDPK的组织和亚细胞定位。然后，使用转基因技术得到杨树候选CDPK的RNAi和过表达突变体，同时将候选CDPK过表达于拟南芥中，对上述突变体进行表型分析，明确候选CDPK蛋白的功能。最后，阐明CDPK调控杨树适应逆境的机制，使用酵母双杂交技术筛选候选CDPK的互作蛋白和下游底物，使用体外磷酸化、免疫共沉淀、双分子荧光互补、烟草LCI技术验证候选CDPK与底物的互作。

土壤盐渍化、重金属污染和低温胁迫为农林业生产带来巨大经济损失。当植物遭受环境胁迫时，会造成体内生理代谢失衡，并引起次级毒害。通过研究分子水平和生理水平上植物的抗逆机制，将为植物的良种选育提供理论基础。本项目以拟南芥（Arabidopsis thaliana）和耐盐木本植物胡杨（Populus euphratica）为实验材料，重点研究拟南芥钙依赖型蛋白激酶CPK27在植物盐胁迫信号转导过程中的功能；H2S在缓解胡杨镉胁迫过程中的调节作用；液泡Ca2+和胞外ATP信号分子在调节胡杨耐盐过程中的功能。拟南芥CPK27是一个细胞膜定位的蛋白激酶，CPK27的表达受到NaCl的诱导，CPK27有可能通过调节活性氧和离子平衡，正向调节盐胁迫信号转导过程。H2S能够明显缓解镉胁迫诱导的胡杨细胞程序化死亡过程。镉胁迫条件下，H2S能够提高抗氧化酶的活性。H2S通过抑制镉离子的内流、将镉离子区隔化到液泡中来缓解镉胁迫。胡杨细胞在盐胁迫处理下，能够迅速将液泡中的Ca2+释放到细胞质中，NaCl诱导的液泡Ca2+的释放过程，受到胞外ATP、H2O2和NO的调节。在胡杨和灰杨中，建立了高效的病毒诱导基因沉默体系（VIGS）。使用烟草脆裂病毒载体能够有效干扰PDS基因的表达，并且VIGS的沉默效应受到温度的调节。使用VIGS技术阐明了胡杨HSF转录因子通过调节WRKY1基因的表达来提高植物耐盐性的机制。阐明了胡杨PeAPY2基因参与调节植物对低温胁迫的响应过程，过表达PeAPY2基因提高了转基因拟南芥抵御低温的能力。PeAPY2通过调节ATP的水平、促进囊泡转运、提高植物细胞膜的修复能力增强了植株抗低温的能力。上述研究成果发表在Gene、Plant Physiology and Biochemistry、Cell Calcium、Plant Science、Tree Physiology、Trees和Plant Physiology杂志上。