【作者】 孙宁；

【导师】 魏凤菊； 王冬梅；

【作者基本信息】 河北农业大学 ， 细胞生物学， 2013， 硕士

【摘要】 Ca²⁺作为植物信号转导途径中重要的第二信使，几乎介导了植物生长发育和对逆境响应的所有反应。CDPKs（或CPKs）即钙离子依赖的蛋白激酶，是一类重要的钙离子信号感受器，在响应逆境胁迫过程中发挥着重要的作用。拟南芥基因组共编码34个CDPKs，该家族成员具较高的序列同源性和典型的结构特征，可能存在功能相似或冗余的现象。实验室前期的工作表明，拟南芥CPK10基因的T-DNA插入突变体cpk10成苗阶段在干旱胁迫下表现出较野生型敏感的表型；而在种子萌发阶段和幼苗时期没有表现出明显逆境相关（ABA、Mannitol、Sorbitol）的生理表型。promoter::GUS组织表达分析显示CPK10在萌发两天的种子及生长十天的幼苗中有较强表达。CPK30与CPK10基因具有高度的序列相似性，推测其与CPK10可能存在功能相似或冗余的现象。为进一步研究CPK10在响应非生物逆境过程中的分子功能，我们拟通过构建cpk10×cpk30双突变纯合体，结合生理表型及逆境相关基因检测，进一步探讨CPK10可能的作用机制。本文构建了cpk10×cpk30双突变纯合体，并对双突变体在种子萌发时期、幼苗时期、成苗时期是否响应干旱、高盐、激素等非生物逆境胁迫过程进行了表型观察。在不同的逆境处理条件下，种子萌发和幼苗期均未发现双突变体表现出区别于野生型的明显表型；成苗期cpk10×cpk30双突变并未表现出比单突变体cpk10更加对干旱敏感的表型；盐处理结果显示，双突变体表现出较单突变体更敏感的表型。为进一步明确CPK10、CPK30可能参与的信号转导过程，通过选取响应非生物逆境不同信号途径（ABA合成、ABA信号等）的标记基因作为目的基因，通过分析双突变体、单突变体及野生型材料在干旱处理条件下，各标记基因转录水平表达量变化，探讨CPK10基因响应干旱逆境胁迫过程中发挥功能的分子机制。试验结果显示，RD29A表现出完全不同于野生型及单突变体的表达趋势变化，表明CPK10/CPK30与水分胁迫响应相关。ABA刺激响应蛋白激酶OST1的变化趋势在野生型、单突变体与双突变体中也存在明显的差异。根据以上结果，推断CPK10与CPK30可能共同参与了依赖ABA的干旱逆境信号转导过程，并且基因间存在功能冗余。更多还原

【Abstract】 Ca²⁺, as an important second messenger in plant signal transduction pathways, almostmediated plant growth and development, and response to adversity reaction. CDPKs（CPKs, the calcium-dependent protein kinase） is an important set of calcium signalingreceptors, and plays crucial role in the process of response to abiotic stresses. Arabidopsisgenome encodes34the CDPKs, members of the family having high sequence homologyand the typical structural features. There may be function similar, or redundancyphenomenon between the members of the family.The laboratory preliminary work shows that the Arabidopsis CPK10gene T-DNAinsertion mutant, cpk10, is more sensitive to drought stress in seedling stage than thewild-type phenotype. In seed germination stage and seedling period cpk10did not showsignificant physiological phenotype with the treatmen of ABA, Mannitol, Sorbitol.Promoter::GUS tissue expression analysis showed strong expression of CPK10in thegermination of seeds in two days and ten days growth seeds. CPK30gene has a highdegree of sequence similarity with CPK10. It speculated that there may be functionalsimilarity or redundant between CPK10and CPK30. For further study of the molecularfunction of CPK10in response to abiotic stress process, we intend to constructedcpk10×cpk30double mutant homozygotes, combined with physiological phenotypes andstress-related gene detection, to further explore the molecular mechanism of CPK10inresponse to abiotic stress process.In this paper, we constructed cpk10×cpk30double mutant and observed the phenotypeof double mutant during the seed germination, seedling period, the seedling period inresponse to abiotic stresses of drought, high salt, hormones and other stress process.Onseed germination and seedling stage, we were not found double mutants exhibitsignificantly different from the wild-type phenotype in different treatment conditions ofadversity. In the seedling period, cpk10×cpk30double mutant did not show more than thesingle mutants cpk10drought-sensitive phenotype; salt treatment showed double mutantsexhibit more sensitive than the single mutant phenotype. Based on the the phenotypeanalysis of the experimental results, we speculated that CPK10, CPK30whether involvedin drought stress of a signal transduction process maturity? Different signaling pathways（ABA synthesis, ABA signal, etc.） marker genes under drought stress are selected as the target gene. By analyzing changes in expression levels of each marker gene transcriptionlevel in the double mutant, single mutants and wild-type materials under drought stressconditions, analysis CPK10gene function in response to the drought stress. RD29Ashowed completely different expression trends in double mutant, wild-type and singlemutants, indicating that CPK10/CPK30involved in the signaling pathway which responseto water stress. ABA stimulated response protein kinase OST1trends, there are alsosignificant differences in wild-type, single mutants and double mutant. Based on the aboveresults, it inferred CPK10and CPK30may jointly involved in the ABA-dependent droughtstress signal transduction. And there are gene functional redundancy between the CPK10and CPK30.更多还原