本文選題：小麥 + CBL-CIPK　； 參考：《海南大學》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并通過多種信號轉(zhuǎn)導途徑來產(chǎn)生不同形式的響應。鈣(Ca2+)信號傳導就是在植物中轉(zhuǎn)導大量刺激或信號的一個非常重要的途徑。Ca2+通過參與調(diào)節(jié)各類鈣解碼器以介導植物中的信號傳導。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合物并在轉(zhuǎn)導這些信號中發(fā)揮非常重要的作用。這兩個基因家族參與了多種刺激-反應耦合的信號網(wǎng)絡通路。盡管CBL-CIPK網(wǎng)絡已經(jīng)證明能夠在植物發(fā)育和各種環(huán)境脅迫的反應中起關鍵作用,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,對其編碼的蛋白進行生物信息學分析。利用qRT-PCR技術分析TaCBL1基因在不同非生物脅迫下的表達模式,通過農(nóng)桿菌介導法將TaCBL1基因轉(zhuǎn)入煙草中,觀察其對非生物脅迫的響應,測定其形態(tài)指標、生理生化指標等,揭示了過表達TaCBL1在非生物脅迫高鹽處理下對煙草的生長發(fā)育的影響,主要的研究結果如下:1.通過對小麥進行不同的非生物脅迫(鹽,低溫,干旱,ABA)處理結果表明,TaCBL1基因在 PEG-6000(20%)、高鹽(200 mM NaCl),低溫(4℃)和 ABA(100μuM)處理的情況下,其表達量會隨著時間的延長發(fā)生變化,其中TaCBL1基因在PEG模擬干旱處理時,發(fā)現(xiàn)3h時根中TaCBL1基因顯著下降,之后慢慢升高,葉片中該基因表達在9h達到最低;200 mM鹽處理下TaCBL1的表達水平一直較低,12h會出現(xiàn)瞬時性的增加之后又降低到低水平;低溫(4℃)處理時,葉片中:TaaCBL/基因的表達量都較低,根中的表達量會有波動,但總體仍是低于Oh的表達量;ABA處理時,TaCBL1基因在葉片和根中的表達量都是逐漸降低的,且根中該基因的表達下降的更快。2.將TaCBL1基因構建到pCAMB1A1300載體上,在煙草原生質(zhì)體中表達TaCBL1蛋白做定位分析,表明TaCBL1蛋白定位在細胞膜上,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)為對鹽是敏感的,這與前人在擬南芥上AtCBL1表現(xiàn)出對鹽的抗逆性不同,當然我們了解到前人在對楊樹PeCBL1基因進行研究時也有類似的表型。4.為了進一步探究TaCBL1沿轉(zhuǎn)入煙草為何會出現(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定,結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，

本文編號：2063849