国家自然科学基金结题报告

项目批准号31101022申请代码C0709归口管理部门收件日期

国家自然科学基金

资助项目结题报告

资助类别：青年科学基金项目

亚类说明：

附注说明：

项目名称：CPK10基因调控拟南芥响应干旱胁迫的分子机制研究

负 责 人：魏凤菊电话：0312-7528249

电子邮件：weifj98@gmail.com

依托单位：河北农业大学

联 系 人：张永升电话：0312-7526131

资助金额：22（万元）累计拨款：22.0（万元）

执行年限：2012.01-2014.12

2015年01月16日填表日期：

国家自然科学基金委员会制（2012年）NSFC-REPORT-2014

Version：1.022.799项目摘要

中文摘要(500字以内)：钙依赖型蛋白激酶（CDPKs或CPKs）是钙信号的重要传递体，拟南芥基因组编码34个CDPKs，其中大部分基因的功能及其参与的信号转导途径仍不清楚。申请人前期研究结果表明，拟南芥CPK10功能缺失突变体表现对干旱显著敏感表型，组织表达分析和气孔开度实验证明CPK10参与了气孔开放与关闭运动过程，这些结果初步表明CPK10通过调节气孔运动参与响应干旱胁迫的信号转导过程。本项目将在前期工作基础上，通过酵母双杂交筛选保卫细胞cDNA文库以及利用串联亲和纯化（TAP）技术寻找CPK10的互作靶蛋白，结合运用遗传学、蛋白质生物化学、细胞生物学等方法与技术，分析研究CPK10在体内参与调控气孔运动的作用，深入探讨拟南芥CPK10蛋白响应干旱信号转导过程的分子机制。此研究为进一步阐明植物的抗旱机制、为利用基因工程手段培育优良的植物抗旱新品种提供理论依据。

拟南芥； 蛋白激酶； 干旱胁迫； AtCPK10；关键词：

Abstract (limited to 1500 characters)：Calcium-dependent protein kinases (CDPKs) functions as important calciumsensors in plants and there are 34 CDPKs in Arabidopsis genome alone. Thebiological functions of most CDPKs in stress signaling remain unclear. Theapplicant's previous results showed the CPK10 mutant exhibited more sensitivephenotypes in response to drought stress than wild type. Expression analysisand stomatal aperture assays demonstrated that CPK10 were involved instomatal opening and closure movement. These data presented that CPK10,possibly via regulating stomatal movements, plays important roles in responseto drought stress. Based on the previous results, the further study in thisproject focused on identifying target protein(s) of CPK10 and characterizingthe function of CPK10 in response to drought stress. Yeast-two hybridinteraction screening from guard cell cDNA libraries and tandem affinitypurification (TAP) were used to identified CPKA10-interacting protein(s), andgenetics, biochemistry and cytobilolgy methods were followed to analysis thesubstrates functions. These rusults will further facilitate more detailinvestigations into plant respons to drought stress, and provided theoriesfor breeding excellent drought-resistant lines.

Arabidopsis; protein kinase; drought stress; AtCPK10; Keywords: NSFC-REPORT-2014

第 1 页国家自然科学基金资助项目结题报告结题摘要

中文摘要(对项目的完成情况及取得成果做简单概述，1000字以内)：拟南芥CPK10参与了干旱胁迫过程中对气孔运动的调节，本课题通过串联亲和纯化（TAP）技术寻找AtCPK10的互作靶蛋白，结合运用生物信息学、蛋白质生物化学、细胞生物学等方法与技术，对AtCPK10参与调控气孔运动的分子机制进行了初步研究。首先通过构建带有双标签（Flag和HA）的pCM1307-Flag-HA-AtCPK10双元表达载体，以农杆菌介导转化拟南芥野生型，筛选获得稳定表达的转基因T3代阳性材料；进而利用TAP技术筛选AtCPK10的互作蛋白，经质谱分析，获得316个蛋白，经生物信息学分析，挑选出9个感兴趣成员。通过RT-PCR方法分析在干旱诱导条件下候选基因转录水平的表达情况，结果显示CPK8、CAT3、HSC70-1受干旱诱导表达量明显增高，推测这些成员可能参与干旱逆境。蛋白质生化及细胞生物学实验证实CPK10和它们之间直接互作的工作正在进行。综合上述结果，本研究为探讨CPK10调节气孔运动的机制奠定了坚实的工作基础。同时对于认识CDPK家族成员之间的关系提供了重要的启示性线索。项目资助发表核心论文1篇，待发表两篇。培养硕士生3名，其中1名已经取得硕士学位，2名在读。项目投入经费22万元，支出19.6858万元，各项支出基本与预算相符。剩余经费2.3142万元，剩余经费计划用于本项目研究后续支出。

TAP技术； AtCPK10； 互作蛋白； 表达变化； 逆境分析关键词：

Abstract (limited to 3000 characters)：• Arabidopsis thaliana Ca2+-dependent protein kinase10 (CPK10) was involvedin regulation of stomatal movments in response to drought stress. Usingtandem affinity purification (TAP) to screen interacting proteins. Combinedwith bioinformatics, protein biochemistry and cytobiology methods, thepreliminary functional characterization of AtCPK10 in regulating stomatalmovements were researched in this investigation. Firstly, the cDNA of CPK10was cloned into vector pCM1307-N-Flag and transferred into Arabidopsis plants(wild type), T3 homozygous lines were obtained. And then, TAP methods wereapplied to screen candidate proteins that interact with CPK10. About 348expressed proteins were obtained from the mass spectrometry(MS) data and 9were selected with candidate proteins. RT-PCR analysis showed thatCPK8、CAT3、HSC70-1 were highly expressed in response to drought stress, thisresult demonstrated that these candidate proteins possibly involved indrought stress. The above results not only lay a solid foundation forelucidating the regulatory mechanism of AtCPK10 on stomatal movements butalso provides insight into understanding the relationship between CDPKsmembers. There are 1 paper was published under the support of NSFC and twopapers would have to wait to be published. One students have obtained masterdegree and two are pursuing. Total input fund was 220,000 yuan. Totalexpenditure was 196,686yuan. The surplus will be continue to spend on thisresearch.NSFC-REPORT-2014

第 2 页国家自然科学基金资助项目结题报告

TAP technology; AtCPK10; interacting proteins; expressionKeywords: analysis; stress analysis

NSFC-REPORT-2014

第 3 页国家自然科学基金资助项目结题报告报告正文

一、研究计划要点及执行情况概述。

研究基本按照原计划执行，一些研究内容稍作调整。（1）检测了CPK10与HSP1蛋白的可能互作方式。通过酵母双杂交技术得出HSP1可以发生自身互作。在利用TAP（串联亲和纯化）技术结合质谱技术分析HSP1与CPK10的作用方式过程中，纯化获得的杂蛋白信号较强，试验未能顺利进行下去。（2）筛选了与CPK10可能互作的其它靶蛋白。分离保卫细胞并建立酵母cDNA文库，通过酵母双杂交技术筛选互作蛋白，获得的蛋白经测序分析多为持家基因表达产物，利用构建的保卫细胞酵母文库筛选CPK10互作蛋白的方法暂告一段。利用串联亲和纯化技术筛选CPK10可能的互作蛋白，再结合质谱分析技术测得蛋白的氨基酸序列，获得多个候选基因。（3）对靶基因是否参与干旱逆境进行了初步分析。检测靶基因受干旱诱导后的表达情况，组织及亚细胞表达情况。（4）初步检测了cpk10突变体材料中靶基因的表达情况，明确的试验结果有待重复。（5）获得了CPK10与CPK30的双突变体，初步进行了基因功能分析。