小麦CIPK基因家族鉴定与分析

发布时间：2018-04-28 01:59

  本文选题：CIPK + 表达谱　； 参考：《华中科技大学》2016年博士论文

【摘要】：干旱、高盐、低温等不利环境因素严重影响植物的正常生长发育。为应对这些不利环境因素,植物在长期的进化过程中形成了一系列响应机制,其中钙离子作为第二信使,在植物响应非生物逆境胁迫的信号传导过程中发挥重要作用。钙调磷酸酶-B类似蛋白(Calcineurin B-like protein, CBL)是植物特有钙离子识别受体蛋白,通过与其互作的激酶蛋白(CBL-interacting protein kinase, CIPK)作用激活后者,进而磷酸化修饰下游靶分子,启动下游一系列响应机制。在模式植物拟南芥中,有关CIPK在响应非生物逆境胁迫中功能的研究报道较多。然而,在重要的粮食作物小麦中,关于CIPK基因的鉴定工作进展缓慢,功能分析也少有报道。小麦具有异源六倍体基因组,其基因组相对于二倍体植物更加庞大、复杂。虽然目前小麦基因组测序工作取得了重大进展,但是还不能提供完整的小麦基因组序列和物理图谱。本研究采用了多种分析方法,对小麦CIPK全基因家族进行了鉴定,共发现79个TaCIPK基因,归属为29个基因簇,并最终从这些基因簇中克隆到20个代表性的基因。在此基础上,我们系统分析了CIPK基因的组织特异性、逆境响应表达谱；研究了TaCIPK与TaCBL的相互作用关系；分析了TaCIPK24在转基因拟南芥中对高盐胁迫响应中的作用；利用基因枪法获得过表达TaCIPK10和TaCIPK10-RNAi的转基因小麦植株；并利用生物信息学方法对小麦CIPK在种子萌发和成熟花粉中的功能进行预测。主要研究结果如下：1)利用生物信息学方法在小麦基因组中成功鉴定了79个TaCIPK基因,将其分为29个基因簇,每个基因簇中的基因为异源基因组染色体上的等位基因,序列高度相似。基因结构分析结果显示,42个TaCIPK基因可以鉴定到完整的结构信息,其中23个基因不含内含子结构,3个基因含有一个内含子,其余16个基因含有5-14个内含子。通过比较乌拉尔图小麦、粗山羊草与小麦相对应的亚基因组CIPK基因序列,结果发现在相应的CIPK基因序列区域间存在高度相似性,但也有一定的差异,现阶段的小麦基因组数据还不能确定CIPK基因家族在六倍体化过程中是否发生了基因丢失或复制现象。根据EST拼接结果和基因组中CIPK的序列,设计特异引物,从小麦中共克隆出20个TaCIPK基因。2)利用半定量RT-PCR技术分析了17个TaCIPK基因在10种小麦组织中的表达情况;鉴定了23个TaCIPK基因对应的探针,并利用R语言从公布的小麦基因芯片数据中分析这23个TaCIPK基因在7种组织中的表达情况。结果显示,TaCIPK家族基因在各个组织或时期中的表达量有所不同,反映了它们可能广泛参与了小麦的生长发育过程。利用基因芯片数据进一步分析了23个TaCIPK基因在种子萌发过程的基因表达模式,发现8个TaCIPK基因在种子萌发过程表达量有显著变化,进一步利用半定量RT-PCR技术对这一结果进行了确证。在花粉发育过程中,不同TaCIPK基因的表达模式也存在较大差异,其中6个TaCIPK基因只在成熟花粉中有高量表达,暗示它们可能在花粉萌发或者花粉管伸长过程中发挥功能。利用半定量RT-PCR技术分析了17个TaCIPK基因在ABA, GA, MeJA, ACC,低温,PEG, H2O2, NaCl和高温处理后小麦根和叶中的表达模式,并随机选取5个基因(TaCIPK7,TaCIPK15, TaCIPK24, TaCIPK31和TaCIPK32),利用实时荧光定量RT-PCR法分析了它们响应ABA, PEG,低温,H2O2和NaCl处理的表达模式。结果表明,不同的TaCIPK基因可以不同程度地对激素信号和非生物逆境胁迫进行响应。3)利用酵母双杂交方法分析鉴定了7个TaCBL与20个TaCIPK蛋白相互作用模式,从中随机选择了7个组合利用双分子荧光互补法在烟草表皮细胞中进行验证。将TaCIPK1蛋白C-端序列进行系列删除(保留NAF结构域),结果显示,删除突变体与TaCBL的结合模式发生变化,说明TaCIPK蛋白C-端除NAF模体外的序列也对与TaCBL的结合发挥作用。我们认为这些侧翼序列主要是通过改变C-端空间构象影响与TaCBL的结合,并提出了CBL-CIPK结合的“凹凸”模型。根据此模型推断,小麦亚基因组上TaCIPK等位基因在C-端的序列的变异可能导致与TaCBL的结合的改变,并利用TaCIPK17-A和TaCIPK-17B1分别与TaCBL的相互作用验证了这一推论。这些结果说明小麦CBL-CIPK调控网络的复杂性,为CBL与CIPK的结合机制研究提供了参考。4)发现在拟南芥中过表达TaCIPK24可以增强拟南芥转基因植株对高盐胁迫的耐受能力。测量了盐处理后拟南芥植株中Na+和K+含量,结果显示,K+含量在转基因和对照组中没有明显差异,而转基因植株明显比对照积累更少的Na+。TaCIPK24可能是通过激活拟南芥的SOS途径,促进排出更多的Na+从而减少过量Na+对拟南芥植株造成的损害。我们发现与对照相比,TaCIPK24过表达的转基因拟南芥可以积累更少的活性氧物质。进一步测定过氧化氢酶、过氧化物酶和超氧化物歧化酶的酶活力的结果显示,转基因植株可能还激活了过氧化物清除系统,从而降低高盐胁迫造成的氧化损伤。5)利用加权共表达网络分析法,构建了小麦种子萌发和花粉发育过程中TaCIPK基因相关的共表达网络。基因功能富集分析结果显示,8个TaCIPK基因在种子萌发时期参与到了碳水化合物代谢、脂类代谢和核小体组装等过程；6个在成熟花粉中特异性高量表达的TaCIPK基因参与到了应激反应、糖代谢、氮代谢、电子传递和离子转运过程。6)组织表达分析发现,TaCIPK10基因在叶片中的表达量明显高于其它组织,使用皮尔森相关系数法筛选到的65个基因与TaCIPK10表现出相同的表达模式；没有发现除TaCIPK10基因外的其它TaCIPK基因与TaCIPK10有相似表达模式,且其它CIPK基因存在功能互补的可能性较小,暗示着TaCIPK10可能发挥着比较特殊的功能。此外我们发现TaCIPK10可以被多种激素和非生物逆境胁迫诱导表达。为研究TaCIPK10的功能,我们构建了TaCIPK10过表达和TaCIPK10-RNAi载体,利用基因枪法转化中国春小麦,获得了8个TaCIPK10过表达转基因小麦株系和9个TaCIPK10-RNAi转基因小麦株系,为后续功能研究工作奠定了基础。
[Abstract]:Adverse environmental factors, such as drought, high salt and low temperature, seriously affect the normal growth and development of plants. In response to these adverse environmental factors, a series of response mechanisms have been formed in the long-term evolution of plants. Calcium ions, as second messengers, play an important role in the signal transduction of plant responses to abiotic stress. Phosphatase -B similar protein (Calcineurin B-like protein, CBL) is a plant specific calcium recognition receptor protein that activates the latter by its interaction with CBL-interacting protein kinase, CIPK, and then phosphorylates downstream target molecules and starts a series of response mechanisms. In model plant Arabidopsis, CIPK. There are many reports on function in response to abiotic stress. However, in the important grain crop wheat, the identification of the CIPK gene is progressing slowly and the functional analysis is rarely reported. The genome of wheat with a heterologous six ploidy genome is larger and complex than the diploid plant. The sequence work has made great progress, but the complete genome sequence and physical map of the wheat have not been provided. A variety of analysis methods have been used to identify the CIPK whole gene family of wheat, and 79 TaCIPK genes have been found, belonging to 29 gene clusters, and 20 representative genes are cloned from these gene clusters. On the basis of this, we systematically analyzed the tissue specificity of CIPK gene, stress response expression spectrum, and studied the interaction between TaCIPK and TaCBL, analyzed the role of TaCIPK24 in the response to high salt stress in transgenic Arabidopsis, and obtained transgenic wheat plants expressing TaCIPK10 and TaCIPK10-RNAi by using the method of gene shooting; and The function of wheat CIPK in Seed Germination and mature pollen was predicted by bioinformatics. The main results were as follows: 1) 79 TaCIPK genes were successfully identified by bioinformatics in the wheat genome, which were divided into 29 gene clusters, and each gene cluster was the allele on the heterologous genome. Genetic analysis showed that 42 TaCIPK genes could identify complete structural information, including 23 genes without intron structure, 3 genes containing one intron, and 16 other 16 genes containing 5-14 introns. By comparing Ural map wheat, the subgenomic CIPK corresponding to wheat was compared with wheat. It is found that there is a high similarity between the corresponding CIPK gene sequences, but there are some differences. The present stage of wheat genome data can not determine whether the CIPK gene family has the phenomenon of gene loss or replication in the process of six ploidy. According to the results of EST splicing and the sequence of CIPK in the genome, a special design is designed. Different primers, 20 TaCIPK genes.2 from wheat were cloned from wheat. The expression of 17 TaCIPK genes in 10 wheat tissues was analyzed by semi quantitative RT-PCR, and 23 TaCIPK genes were identified, and the expression of the 23 TaCIPK genes in 7 tissues was analyzed from the published wheat gene chip data. The results show that the expression of TaCIPK family genes in various tissues and periods is different, reflecting that they may be widely involved in the growth and development of wheat. By using gene chip data, the gene expression patterns of 23 TaCIPK genes in seed germination process are further analyzed, and 8 TaCIPK genes are found in the seed germination process table. The results were confirmed by semi quantitative RT-PCR. In the process of pollen development, the expression patterns of different TaCIPK genes were also different. 6 TaCIPK genes were expressed in mature pollen, suggesting that they may play a role in pollen germination or pollen tube elongation. Function. Semi quantitative RT-PCR technique was used to analyze the expression patterns of 17 TaCIPK genes in ABA, GA, MeJA, ACC, low temperature, PEG, H2O2, NaCl and high temperature treatment of wheat roots and leaves, and randomly selected 5 genes (TaCIPK7, TaCIPK15, TaCIPK24, etc.). The expression patterns of temperature, H2O2 and NaCl treatment. The results show that different TaCIPK genes can respond to hormone signal and abiotic stress in varying degrees to.3). The interaction patterns of 7 TaCBL and 20 TaCIPK proteins are identified by yeast two hybrid method, and 7 combinations using double molecular fluorescence complementary method are selected from the machine. The C- terminal sequence of TaCIPK1 protein was deleted from the tobacco epidermal cells (NAF domain). The result showed that the binding mode of the deletion mutant and TaCBL changed, indicating that the sequence of the TaCIPK protein C- end except the NAF module also played a role in the binding with TaCBL. The C- terminal space conformation affects the combination of TaCBL and the "concave and convex" model of CBL-CIPK binding. According to this model, it is concluded that the mutation of the TaCIPK allele in the C- terminal of the wheat subgenome may lead to the change of the binding with TaCBL, and the interaction of TaCIPK17-A and TaCIPK-17B1 with TaCBL, respectively, is used to verify this push. These results illustrate the complexity of the wheat CBL-CIPK regulatory network and provide a reference.4 for the study of the combination mechanism of CBL and CIPK.) it was found that overexpression of TaCIPK24 in Arabidopsis could enhance the tolerance to high salt stress in transgenic plants of Arabidopsis. The content of Na+ and K+ in Arabidopsis plants after salt treatment was measured. The results showed that the content of K+ was in turn. There is no significant difference between the gene and the control group, while the genetically modified plants obviously accumulate less Na+.TaCIPK24 than the control. It may be by activating the SOS pathway of Arabidopsis thaliana to promote the excretion of more Na+ and reduce the damage caused by excessive Na+ to Arabidopsis plants. We found that the transgenic Arabidopsis of TaCIPK24 over expression can be accumulated compared with the control. The results of further determination of the activity of catalase, peroxidase and superoxide dismutase showed that the transgenic plants may also activate the peroxidase scavenging system, thus reducing the oxidative damage.5 caused by high salt stress, and using the weighted co expression network analysis to construct the germination of wheat seeds. TaCIPK gene related co expression network in the process of pollen development. The results of gene function enrichment analysis showed that 8 TaCIPK genes participated in carbohydrate metabolism, lipid metabolism and nucleosome assembly during the germination period, and 6 TaCIPK genes with high specific expression in mature pollen participated in the stress response and sugar generation. Tissue expression analysis of nitrogen metabolism, electron transfer and ion transport process.6 found that the expression of TaCIPK10 gene in leaves was significantly higher than that of other tissues. The 65 genes screened by Pearson correlation coefficient showed the same expression pattern with TaCIPK10, and other TaCIPK genes except TaCIPK10 gene and TaCIPK10 were not found. There is a similar expression pattern, and other CIPK genes are less likely to complement each other, suggesting that TaCIPK10 may play a more specific function. In addition, we found that TaCIPK10 can be induced by a variety of hormones and abiotic stress. In order to study the function of TaCIPK10, we have constructed the TaCIPK10 overexpression and TaCIPK10-RNAi vector. 8 TaCIPK10 overexpressed transgenic wheat lines and 9 TaCIPK10-RNAi transgenic wheat lines were obtained by using the gene shot method to transform spring wheat in China, which laid the foundation for the follow-up function research.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1;Q943.2

【相似文献】

相关会议论文 前2条

1 刘佳;吕应堂;;水稻CIPK基因的分离与鉴定[A];湖北省植物生理学会第十五次学术研讨会论文集[C];2007年

2 郭军;张洪;丁可;段迎辉;黄丽丽;康振生;;小麦CIPK基因的克隆、表达特征及功能分析[A];中国植物病理学会2009年学术年会论文集[C];2009年

相关博士学位论文 前1条

1 孙涛;小麦CIPK基因家族鉴定与分析[D];华中科技大学;2016年

，

本文编号：1813282