發(fā)布時(shí)間：2018-04-28 01:59

  本文選題：CIPK + 表達(dá)譜　； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：干旱、高鹽、低溫等不利環(huán)境因素嚴(yán)重影響植物的正常生長發(fā)育。為應(yīng)對這些不利環(huán)境因素,植物在長期的進(jìn)化過程中形成了一系列響應(yīng)機(jī)制,其中鈣離子作為第二信使,在植物響應(yīng)非生物逆境脅迫的信號(hào)傳導(dǎo)過程中發(fā)揮重要作用。鈣調(diào)磷酸酶-B類似蛋白(Calcineurin B-like protein, CBL)是植物特有鈣離子識(shí)別受體蛋白,通過與其互作的激酶蛋白(CBL-interacting protein kinase, CIPK)作用激活后者,進(jìn)而磷酸化修飾下游靶分子,啟動(dòng)下游一系列響應(yīng)機(jī)制。在模式植物擬南芥中,有關(guān)CIPK在響應(yīng)非生物逆境脅迫中功能的研究報(bào)道較多。然而,在重要的糧食作物小麥中,關(guān)于CIPK基因的鑒定工作進(jìn)展緩慢,功能分析也少有報(bào)道。小麥具有異源六倍體基因組,其基因組相對于二倍體植物更加龐大、復(fù)雜。雖然目前小麥基因組測序工作取得了重大進(jìn)展,但是還不能提供完整的小麥基因組序列和物理圖譜。本研究采用了多種分析方法,對小麥CIPK全基因家族進(jìn)行了鑒定,共發(fā)現(xiàn)79個(gè)TaCIPK基因,歸屬為29個(gè)基因簇,并最終從這些基因簇中克隆到20個(gè)代表性的基因。在此基礎(chǔ)上,我們系統(tǒng)分析了CIPK基因的組織特異性、逆境響應(yīng)表達(dá)譜；研究了TaCIPK與TaCBL的相互作用關(guān)系；分析了TaCIPK24在轉(zhuǎn)基因擬南芥中對高鹽脅迫響應(yīng)中的作用；利用基因槍法獲得過表達(dá)TaCIPK10和TaCIPK10-RNAi的轉(zhuǎn)基因小麥植株；并利用生物信息學(xué)方法對小麥CIPK在種子萌發(fā)和成熟花粉中的功能進(jìn)行預(yù)測。主要研究結(jié)果如下：1)利用生物信息學(xué)方法在小麥基因組中成功鑒定了79個(gè)TaCIPK基因,將其分為29個(gè)基因簇,每個(gè)基因簇中的基因?yàn)楫愒椿蚪M染色體上的等位基因,序列高度相似�；蚪Y(jié)構(gòu)分析結(jié)果顯示,42個(gè)TaCIPK基因可以鑒定到完整的結(jié)構(gòu)信息,其中23個(gè)基因不含內(nèi)含子結(jié)構(gòu),3個(gè)基因含有一個(gè)內(nèi)含子,其余16個(gè)基因含有5-14個(gè)內(nèi)含子。通過比較烏拉爾圖小麥、粗山羊草與小麥相對應(yīng)的亞基因組CIPK基因序列,結(jié)果發(fā)現(xiàn)在相應(yīng)的CIPK基因序列區(qū)域間存在高度相似性,但也有一定的差異,現(xiàn)階段的小麥基因組數(shù)據(jù)還不能確定CIPK基因家族在六倍體化過程中是否發(fā)生了基因丟失或復(fù)制現(xiàn)象。根據(jù)EST拼接結(jié)果和基因組中CIPK的序列,設(shè)計(jì)特異引物,從小麥中共克隆出20個(gè)TaCIPK基因。2)利用半定量RT-PCR技術(shù)分析了17個(gè)TaCIPK基因在10種小麥組織中的表達(dá)情況;鑒定了23個(gè)TaCIPK基因?qū)?yīng)的探針,并利用R語言從公布的小麥基因芯片數(shù)據(jù)中分析這23個(gè)TaCIPK基因在7種組織中的表達(dá)情況。結(jié)果顯示,TaCIPK家族基因在各個(gè)組織或時(shí)期中的表達(dá)量有所不同,反映了它們可能廣泛參與了小麥的生長發(fā)育過程。利用基因芯片數(shù)據(jù)進(jìn)一步分析了23個(gè)TaCIPK基因在種子萌發(fā)過程的基因表達(dá)模式,發(fā)現(xiàn)8個(gè)TaCIPK基因在種子萌發(fā)過程表達(dá)量有顯著變化,進(jìn)一步利用半定量RT-PCR技術(shù)對這一結(jié)果進(jìn)行了確證。在花粉發(fā)育過程中,不同TaCIPK基因的表達(dá)模式也存在較大差異,其中6個(gè)TaCIPK基因只在成熟花粉中有高量表達(dá),暗示它們可能在花粉萌發(fā)或者花粉管伸長過程中發(fā)揮功能。利用半定量RT-PCR技術(shù)分析了17個(gè)TaCIPK基因在ABA, GA, MeJA, ACC,低溫,PEG, H2O2, NaCl和高溫處理后小麥根和葉中的表達(dá)模式,并隨機(jī)選取5個(gè)基因(TaCIPK7,TaCIPK15, TaCIPK24, TaCIPK31和TaCIPK32),利用實(shí)時(shí)熒光定量RT-PCR法分析了它們響應(yīng)ABA, PEG,低溫,H2O2和NaCl處理的表達(dá)模式。結(jié)果表明,不同的TaCIPK基因可以不同程度地對激素信號(hào)和非生物逆境脅迫進(jìn)行響應(yīng)。3)利用酵母雙雜交方法分析鑒定了7個(gè)TaCBL與20個(gè)TaCIPK蛋白相互作用模式,從中隨機(jī)選擇了7個(gè)組合利用雙分子熒光互補(bǔ)法在煙草表皮細(xì)胞中進(jìn)行驗(yàn)證。將TaCIPK1蛋白C-端序列進(jìn)行系列刪除(保留NAF結(jié)構(gòu)域),結(jié)果顯示,刪除突變體與TaCBL的結(jié)合模式發(fā)生變化,說明TaCIPK蛋白C-端除NAF模體外的序列也對與TaCBL的結(jié)合發(fā)揮作用。我們認(rèn)為這些側(cè)翼序列主要是通過改變C-端空間構(gòu)象影響與TaCBL的結(jié)合,并提出了CBL-CIPK結(jié)合的“凹凸”模型。根據(jù)此模型推斷,小麥亞基因組上TaCIPK等位基因在C-端的序列的變異可能導(dǎo)致與TaCBL的結(jié)合的改變,并利用TaCIPK17-A和TaCIPK-17B1分別與TaCBL的相互作用驗(yàn)證了這一推論。這些結(jié)果說明小麥CBL-CIPK調(diào)控網(wǎng)絡(luò)的復(fù)雜性,為CBL與CIPK的結(jié)合機(jī)制研究提供了參考。4)發(fā)現(xiàn)在擬南芥中過表達(dá)TaCIPK24可以增強(qiáng)擬南芥轉(zhuǎn)基因植株對高鹽脅迫的耐受能力。測量了鹽處理后擬南芥植株中Na+和K+含量,結(jié)果顯示,K+含量在轉(zhuǎn)基因和對照組中沒有明顯差異,而轉(zhuǎn)基因植株明顯比對照積累更少的Na+。TaCIPK24可能是通過激活擬南芥的SOS途徑,促進(jìn)排出更多的Na+從而減少過量Na+對擬南芥植株造成的損害。我們發(fā)現(xiàn)與對照相比,TaCIPK24過表達(dá)的轉(zhuǎn)基因擬南芥可以積累更少的活性氧物質(zhì)。進(jìn)一步測定過氧化氫酶、過氧化物酶和超氧化物歧化酶的酶活力的結(jié)果顯示,轉(zhuǎn)基因植株可能還激活了過氧化物清除系統(tǒng),從而降低高鹽脅迫造成的氧化損傷。5)利用加權(quán)共表達(dá)網(wǎng)絡(luò)分析法,構(gòu)建了小麥種子萌發(fā)和花粉發(fā)育過程中TaCIPK基因相關(guān)的共表達(dá)網(wǎng)絡(luò)�；蚬δ芨患治鼋Y(jié)果顯示,8個(gè)TaCIPK基因在種子萌發(fā)時(shí)期參與到了碳水化合物代謝、脂類代謝和核小體組裝等過程；6個(gè)在成熟花粉中特異性高量表達(dá)的TaCIPK基因參與到了應(yīng)激反應(yīng)、糖代謝、氮代謝、電子傳遞和離子轉(zhuǎn)運(yùn)過程。6)組織表達(dá)分析發(fā)現(xiàn),TaCIPK10基因在葉片中的表達(dá)量明顯高于其它組織,使用皮爾森相關(guān)系數(shù)法篩選到的65個(gè)基因與TaCIPK10表現(xiàn)出相同的表達(dá)模式；沒有發(fā)現(xiàn)除TaCIPK10基因外的其它TaCIPK基因與TaCIPK10有相似表達(dá)模式,且其它CIPK基因存在功能互補(bǔ)的可能性較小,暗示著TaCIPK10可能發(fā)揮著比較特殊的功能。此外我們發(fā)現(xiàn)TaCIPK10可以被多種激素和非生物逆境脅迫誘導(dǎo)表達(dá)。為研究TaCIPK10的功能,我們構(gòu)建了TaCIPK10過表達(dá)和TaCIPK10-RNAi載體,利用基因槍法轉(zhuǎn)化中國春小麥,獲得了8個(gè)TaCIPK10過表達(dá)轉(zhuǎn)基因小麥株系和9個(gè)TaCIPK10-RNAi轉(zhuǎn)基因小麥株系,為后續(xù)功能研究工作奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S512.1;Q943.2

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