本文選題：pag1 + CCRI24��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：干旱是危害農(nóng)業(yè)生產(chǎn)的全球性問題。我國是世界上最大的棉花生產(chǎn)國,且棉花在國民經(jīng)濟和社會發(fā)展中占有重要地位。但是,我國土地面積的一半以上都處于干旱和半干旱地區(qū),而且我國具有人多地少、糧棉爭地矛盾突出的現(xiàn)狀。所以,在“不與糧爭田”的前提下,抗旱棉花新品種的培育和發(fā)展旱地植棉將是我國實現(xiàn)糧棉雙豐收的重要途徑。棉花是目前世界上種植面積最大的抗旱先鋒作物,其在進化上可能具有獨特的抗旱機制。有報道表明激素油菜素甾醇類化合物(BRs,brassinosteroids)不僅在植物的生長發(fā)育方面發(fā)揮著重要作用,而且參與植物的逆境脅迫響應(yīng)。同時,亞洲棉、雷蒙德氏棉和陸地棉的全基因組已測序完成。在此背景下,利用現(xiàn)代分子生物學(xué)技術(shù),研究BRs參與調(diào)控的棉花抗旱機制,克隆棉花新的抗旱相關(guān)基因以培育優(yōu)良的棉花抗旱新品種,對我國棉花產(chǎn)業(yè)的穩(wěn)定發(fā)展具有重要意義。本論文利用本實驗室的一個棉花BRs缺失突變體pag1開展研究,結(jié)果如下:1)首先,利用土培法研究pag1的抗旱性實驗結(jié)果表明pag1對干旱脅迫較為敏感且存活率顯著低于對照CCRI24(中24)。在水培條件下,用6%PEG6000模擬干旱處理上述材料也得到了同樣的結(jié)果,并且用24-epi-BL(24-表-油菜素內(nèi)酯)處理的pag1的存活率顯著高于pag1。2)進一步研究發(fā)現(xiàn),pag1的主根長度和側(cè)根數(shù)目均顯著小于對照CCRI24,并且用24-epi-BL處理pag1可顯著增加其主根長度和側(cè)根數(shù)目。根系生物量的統(tǒng)計發(fā)現(xiàn),無論是干重還是鮮重,pag1的根系生物量均顯著低于對照CCRI24和24-epi-BL處理的pag1。對影響根系發(fā)育的生長素運輸載體Gh PIN2和Gh LAX3的研究發(fā)現(xiàn),他們的基因表達量在pag1中均低于對照CCRI24和24-epi-BL處理的pag1。3)對葉片氣孔的研究發(fā)現(xiàn),pag1的葉片氣孔密度顯著高于對照CCRI24,而使用24-epi-BL噴施處理pag1的葉片可以顯著降低其氣孔密度;氣孔開度研究結(jié)果表明,在ABA處理后pag1的氣孔開度顯著大于對照CCRI24的氣孔開度,但是24-epi-BL處理的pag1葉片的氣孔開度顯著變小。ABA是調(diào)控逆境脅迫重要的植物激素,對其含量測定結(jié)果顯示,pag1中的ABA含量顯著低于對照CCRI24和24-epi-BL處理的pag1。4)對干旱脅迫相關(guān)的基因的表達量檢測結(jié)果顯示,在干旱脅迫條件下,CDPK1,NCED3,ERD10和LEA在CCRI24和24-epi-BL處理pag1中的表達量均顯著高于pag1。雖然ERD1,RD22和RD26只有在CCRI24中的表達量顯著高于pag1,但是在24-epi-BL處理pag1中的表達量也略高于在pag1中的表達量。5)光合作用速率測定結(jié)果顯示,pag1的光合速率顯著低于對照CCRI24和24-epi-BL處理的pag1。生物量統(tǒng)計結(jié)果顯示,無論是干重還是鮮重,pag1的生物量顯著低于對照CCRI24和24-epi-BL處理的pag1。同時,透射電子顯微鏡觀察結(jié)果顯示pag1葉綠體中無淀粉粒的積累,而對照CCRI24和24-epi-BL處理的pag1中均有淀粉粒積累,且葉片淀粉染色也得到同樣的結(jié)果。農(nóng)藝性狀的調(diào)查結(jié)果顯示,無論在正常還是干旱條件下,BRs缺失突變體pag1在株高、果枝數(shù)、桃數(shù)和籽棉產(chǎn)量方面均顯著低于對照CCRI24和24-epi-BL處理pag1。6)為了進一步揭示BRs調(diào)控棉花耐旱性的作用機制,我們采用i TRAQ方法檢測6%PEG6000處理0 h,12 h,24 h,36 h條件下的pag1和CCRI24中的差異蛋白,在pag1/CCRI24±1.2(P value0.05)的篩選條件下共鑒定到2100個差異蛋白,其中上調(diào)蛋白數(shù)目為1132個,下調(diào)蛋白數(shù)目為1105個,上調(diào)和下調(diào)共同擁有蛋白數(shù)目為137個。GO(Gene ontology)分析顯示上調(diào)蛋白主要聚類于細(xì)胞進程、代謝過程和刺激響應(yīng)等12類中,下調(diào)表達的差異蛋白主要聚類于刺激響應(yīng)、大分子復(fù)合物和代謝過程等13類中。在下調(diào)表達的差異蛋白中,有許多是參與干旱脅迫響應(yīng)的,例如ascorbate peroxidase(Cot AD_01708),dehydrin erd10-like protein(Cot AD_58358),nadh dehydrogenase(Cot AD_48405),peroxidase 60(Cot AD_42424),abscisic acid receptor PYR1-like(Cot AD_03959),superoxide dismutase(Cot AD_51044),pyrroline-5-carboxylate reductase(Cot AD_22901)等等。本研究利用棉花BRs缺失突變體pag1,從根系、氣孔、能量合成、基因表達和蛋白組學(xué)等方面全面揭示了BRs缺失造成植物對干旱脅迫敏感的作用機制,使我們認(rèn)識了BRs在生長發(fā)育和干旱脅迫中的重要作用,為我們?nèi)绾卫弥参锛に谺Rs和為我們利用分子生物學(xué)方法克隆BRs相關(guān)基因以培育抗旱性強、高產(chǎn)穩(wěn)產(chǎn)的棉花新品種提供了重要的理論依據(jù)。
[Abstract]:Drought is a global problem endangering agricultural production. China is the largest cotton producer in the world, and cotton plays an important role in the national economy and social development. However, more than half of the land area in China is in arid and semi-arid areas, and there are many people in China, and the contradiction between grain and cotton disputes is prominent. On the premise of "not competing with grain", the cultivation and development of new drought resistant cotton varieties will be an important way to realize the double harvest of grain and cotton in China. Cotton is the largest drought resistant pioneer crop in the world, and it may have a unique mechanism of drought resistance in the evolution. It has been reported that the hormone brassinol compounds (BRs Brassinosteroids) not only plays an important role in the growth and development of plants, but also participates in the stress response of plants. At the same time, the whole genome of Asian cotton, Raymond S cotton and upland cotton has been sequenced. Under this background, using modern molecular biology technology to study the drought resistance mechanism of cotton with the participation of BRs and clone cotton new. The drought resistance related genes are important to cultivate new varieties of drought resistant cotton, which is of great significance to the stable development of cotton industry in China. This paper uses a cotton BRs deletion mutant PAG1 in our laboratory to carry out the study. The results are as follows: 1) first, the results of the study on the drought resistance of PAG1 by soil culture show that PAG1 is more sensitive to drought stress. And the survival rate was significantly lower than that of the control CCRI24 (24). Under the hydroponic condition, the same results were obtained by 6%PEG6000 simulated drought treatment, and the survival rate of PAG1 treated with 24-epi-BL (24- - brassinolide) was significantly higher than that of pag1.2). The length of the main root and the number of lateral roots of PAG1 were significantly smaller than those of the control. CCRI24, and the use of 24-epi-BL to treat PAG1 significantly increased the length of the root and the number of lateral roots. The root biomass of the root system found that both dry weight and fresh weight, the root biomass of PAG1 was significantly lower than that of the CCRI24 and 24-epi-BL treated pag1. on the auxin transport carrier, Gh PIN2 and Gh LAX3, affecting the development of root system. The gene expression in PAG1 was lower than that of the control CCRI24 and 24-epi-BL treated pag1.3). The stomatal density of the leaves of the leaves of PAG1 was significantly higher than that of the control CCRI24, and the leaves of the PAG1 treated with 24-epi-BL spraying could significantly reduce the stomatal density, and the stomatal opening results showed that the stomata of PAG1 was opened after ABA treatment. The degree of stomatal opening was significantly greater than that of the control CCRI24, but the stomatal opening of the 24-epi-BL treated PAG1 leaves was significantly smaller and.ABA was an important plant hormone regulating stress stress. The results of its content determination showed that the content of ABA in PAG1 was significantly lower than that of the control CCRI24 and 24-epi-BL treated pag1.4) and the expression of genes related to drought stress was detected. The results showed that under drought stress, the expressions of CDPK1, NCED3, ERD10 and LEA in CCRI24 and 24-epi-BL were significantly higher than pag1., while RD22 and RD26 were significantly higher than those in CCRI24, but the amount of expression in the PAG1 was slightly higher than that in the PAG1. The results showed that the photosynthetic rate of PAG1 was significantly lower than that of the control CCRI24 and 24-epi-BL, and the statistical results of pag1. biomass showed that the biomass of PAG1 was significantly lower than that of CCRI24 and 24-epi-BL treated pag1., and the results of transmission electron microscopy showed that the accumulation of no starch granules in the PAG1 chloroplasts was observed by the transmission electron microscope. Amyloid accumulation was found in the PAG1 treated PAG1 and 24-epi-BL, and the same results were obtained in the leaf starch staining. The results of the agronomic traits showed that the BRs missing mutant PAG1 was higher in the plant height, the number of fruit branches, the number of peach and the yield of seed cotton were significantly lower than that of the control CCRI24 and 24-epi-BL treated pag1.6. In order to further reveal the mechanism of BRs regulation of Cotton Drought tolerance, we detected the difference protein in PAG1 and CCRI24 under the conditions of 0 h, 12 h, 24 h, 36 h, and 2100 differential proteins under pag1/CCRI24 1.2 (P) conditions, with the I TRAQ method to detect the difference proteins under the conditions of pag1/CCRI24 1.2 (P). The number of up regulation proteins was 1132, down regulated protein. The number of 1105, up and down 137.GO (Gene ontology) analysis showed that up regulated proteins were mainly clustered in the 12 categories of cell process, metabolic process and stimulus response. The differentially expressed proteins were mainly clustered in the 13 categories of stimulus response, macromolecule complex and metabolic process. There are many proteins involved in the response to drought stress, such as ascorbate peroxidase (Cot AD_01708), dehydrin erd10-like protein (Cot AD_58358), NADH dehydrogenase (Cot). Reductase (Cot AD_22901) and so on. This study uses the BRs deletion mutant PAG1 of cotton, from the root, stomata, energy synthesis, gene expression and proteomics, comprehensively reveals the mechanism of BRs deletion causing plant sensitivity to drought stress, which makes us understand the important role of BRs in the growth and drought stress of BRs and how it is for us The use of plant hormone BRs and the cloning of BRs related genes by molecular biological methods provide important theoretical basis for the cultivation of new varieties of cotton with strong drought resistance, high yield and stable yield.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S562

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