本文選題：小麥 + CBL-CIPK��； 參考：《海南大學(xué)》2017年碩士論文

【摘要】：在自然環(huán)境中,植物經(jīng)常受到多種脅迫刺激。這些刺激可以被植物感應(yīng)并通過(guò)多種信號(hào)轉(zhuǎn)導(dǎo)途徑來(lái)產(chǎn)生不同形式的響應(yīng)。鈣(Ca2+)信號(hào)傳導(dǎo)就是在植物中轉(zhuǎn)導(dǎo)大量刺激或信號(hào)的一個(gè)非常重要的途徑。Ca2+通過(guò)參與調(diào)節(jié)各類(lèi)鈣解碼器以介導(dǎo)植物中的信號(hào)傳導(dǎo)。在這些解碼器中,鈣調(diào)磷酸酶蛋白(CalcineurinB-likeproteins,CBL)和與CBL蛋白特異性互作的CIPK蛋白(CBL-interacting protein kinase)。這種激酶蛋白形成復(fù)合物并在轉(zhuǎn)導(dǎo)這些信號(hào)中發(fā)揮非常重要的作用。這兩個(gè)基因家族參與了多種刺激-反應(yīng)耦合的信號(hào)網(wǎng)絡(luò)通路。盡管CBL-CIPK網(wǎng)絡(luò)已經(jīng)證明能夠在植物發(fā)育和各種環(huán)境脅迫的反應(yīng)中起關(guān)鍵作用,但是在小麥中所起到的功能了解甚少。為了解小麥CBL1所起的作用,首先克隆出TaCBL1基因,對(duì)其編碼的蛋白進(jìn)行生物信息學(xué)分析。利用qRT-PCR技術(shù)分析TaCBL1基因在不同非生物脅迫下的表達(dá)模式,通過(guò)農(nóng)桿菌介導(dǎo)法將TaCBL1基因轉(zhuǎn)入煙草中,觀察其對(duì)非生物脅迫的響應(yīng),測(cè)定其形態(tài)指標(biāo)、生理生化指標(biāo)等,揭示了過(guò)表達(dá)TaCBL1在非生物脅迫高鹽處理下對(duì)煙草的生長(zhǎng)發(fā)育的影響,主要的研究結(jié)果如下:1.通過(guò)對(duì)小麥進(jìn)行不同的非生物脅迫(鹽,低溫,干旱,ABA)處理結(jié)果表明,TaCBL1基因在 PEG-6000(20%)、高鹽(200 mM NaCl),低溫(4℃)和 ABA(100μuM)處理的情況下,其表達(dá)量會(huì)隨著時(shí)間的延長(zhǎng)發(fā)生變化,其中TaCBL1基因在PEG模擬干旱處理時(shí),發(fā)現(xiàn)3h時(shí)根中TaCBL1基因顯著下降,之后慢慢升高,葉片中該基因表達(dá)在9h達(dá)到最低;200 mM鹽處理下TaCBL1的表達(dá)水平一直較低,12h會(huì)出現(xiàn)瞬時(shí)性的增加之后又降低到低水平;低溫(4℃)處理時(shí),葉片中:TaaCBL/基因的表達(dá)量都較低,根中的表達(dá)量會(huì)有波動(dòng),但總體仍是低于Oh的表達(dá)量;ABA處理時(shí),TaCBL1基因在葉片和根中的表達(dá)量都是逐漸降低的,且根中該基因的表達(dá)下降的更快。2.將TaCBL1基因構(gòu)建到pCAMB1A1300載體上,在煙草原生質(zhì)體中表達(dá)TaCBL1蛋白做定位分析,表明TaCBL1蛋白定位在細(xì)胞膜上,表明TaCBL1主要在質(zhì)膜上起作用。3.利用農(nóng)桿菌介導(dǎo)法將TaCBL1基因轉(zhuǎn)入煙草中在不同的非生物脅迫下觀察其表型,有趣的是其表現(xiàn)為對(duì)鹽是敏感的,這與前人在擬南芥上AtCBL1表現(xiàn)出對(duì)鹽的抗逆性不同,當(dāng)然我們了解到前人在對(duì)楊樹(shù)PeCBL1基因進(jìn)行研究時(shí)也有類(lèi)似的表型。4.為了進(jìn)一步探究TaCBL1沿轉(zhuǎn)入煙草為何會(huì)出現(xiàn)這種表型,我們?nèi)∞D(zhuǎn)基因煙草在不同濃度鹽處理下的葉片和根部組織,做生理生化分析和鈉鉀離子含量的測(cè)定,結(jié)果表明鹽處理下煙草葉和根中的K+/Na+,野生型都要比轉(zhuǎn)基因煙草的高。
[Abstract]:In the natural environment, plants are often stimulated by a variety of stresses. These stimuli can be induced by plants and through multiple signal transduction pathways to produce different forms of response. Calcium (Ca 2) signal transduction is a very important pathway that transduces a large number of stimuli or signals in plants. [Ca 2] 2 participates in the regulation of various calcium decoders to mediate the signal transduction in plants. In these decoders, the CalcineurinB-like proteinsl and the CBL-interacting protein kinase). This kinase protein forms a complex and plays a very important role in transduction of these signals. These two gene families are involved in multiple stimuli-response coupled signaling network pathways. Although CBL-CIPK networks have been shown to play a key role in plant development and responses to various environmental stresses, little is known about the functions played in wheat. In order to understand the role of CBL1 in wheat, TaCBL1 gene was cloned and its encoded protein was analyzed by bioinformatics. The expression patterns of TaCBL1 gene under different abiotic stress were analyzed by qRT-PCR. TaCBL1 gene was transferred into tobacco by Agrobacterium tumefaciens. The response of TaCBL1 gene to abiotic stress was observed and its morphological indexes, physiological and biochemical indexes were measured. The effects of overexpression of TaCBL1 on the growth and development of tobacco under abiotic stress and high salt stress were revealed. The main results were as follows: 1. The results of different abiotic stress (salt, low temperature, drought abscisic acid) on wheat showed that the expression of TaCBL1 gene changed with time under PEG-6000 (20%), high salt (200mm NaCl), low temperature (4 鈩，

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