利用pag1突变体研究BRs调控棉花抗旱性的作用机制

发布时间：2018-06-28 17:36

本文选题：pag1 + CCRI24　；参考：《华中农业大学》2017年博士论文

【摘要】：干旱是危害农业生产的全球性问题。我国是世界上最大的棉花生产国,且棉花在国民经济和社会发展中占有重要地位。但是,我国土地面积的一半以上都处于干旱和半干旱地区,而且我国具有人多地少、粮棉争地矛盾突出的现状。所以,在“不与粮争田”的前提下,抗旱棉花新品种的培育和发展旱地植棉将是我国实现粮棉双丰收的重要途径。棉花是目前世界上种植面积最大的抗旱先锋作物,其在进化上可能具有独特的抗旱机制。有报道表明激素油菜素甾醇类化合物(BRs,brassinosteroids)不仅在植物的生长发育方面发挥着重要作用,而且参与植物的逆境胁迫响应。同时,亚洲棉、雷蒙德氏棉和陆地棉的全基因组已测序完成。在此背景下,利用现代分子生物学技术,研究BRs参与调控的棉花抗旱机制,克隆棉花新的抗旱相关基因以培育优良的棉花抗旱新品种,对我国棉花产业的稳定发展具有重要意义。本论文利用本实验室的一个棉花BRs缺失突变体pag1开展研究,结果如下:1)首先,利用土培法研究pag1的抗旱性实验结果表明pag1对干旱胁迫较为敏感且存活率显著低于对照CCRI24(中24)。在水培条件下,用6%PEG6000模拟干旱处理上述材料也得到了同样的结果,并且用24-epi-BL(24-表-油菜素内酯)处理的pag1的存活率显著高于pag1。2)进一步研究发现,pag1的主根长度和侧根数目均显著小于对照CCRI24,并且用24-epi-BL处理pag1可显著增加其主根长度和侧根数目。根系生物量的统计发现,无论是干重还是鲜重,pag1的根系生物量均显著低于对照CCRI24和24-epi-BL处理的pag1。对影响根系发育的生长素运输载体Gh PIN2和Gh LAX3的研究发现,他们的基因表达量在pag1中均低于对照CCRI24和24-epi-BL处理的pag1。3)对叶片气孔的研究发现,pag1的叶片气孔密度显著高于对照CCRI24,而使用24-epi-BL喷施处理pag1的叶片可以显著降低其气孔密度;气孔开度研究结果表明,在ABA处理后pag1的气孔开度显著大于对照CCRI24的气孔开度,但是24-epi-BL处理的pag1叶片的气孔开度显著变小。ABA是调控逆境胁迫重要的植物激素,对其含量测定结果显示,pag1中的ABA含量显著低于对照CCRI24和24-epi-BL处理的pag1。4)对干旱胁迫相关的基因的表达量检测结果显示,在干旱胁迫条件下,CDPK1,NCED3,ERD10和LEA在CCRI24和24-epi-BL处理pag1中的表达量均显著高于pag1。虽然ERD1,RD22和RD26只有在CCRI24中的表达量显著高于pag1,但是在24-epi-BL处理pag1中的表达量也略高于在pag1中的表达量。5)光合作用速率测定结果显示,pag1的光合速率显著低于对照CCRI24和24-epi-BL处理的pag1。生物量统计结果显示,无论是干重还是鲜重,pag1的生物量显著低于对照CCRI24和24-epi-BL处理的pag1。同时,透射电子显微镜观察结果显示pag1叶绿体中无淀粉粒的积累,而对照CCRI24和24-epi-BL处理的pag1中均有淀粉粒积累,且叶片淀粉染色也得到同样的结果。农艺性状的调查结果显示,无论在正常还是干旱条件下,BRs缺失突变体pag1在株高、果枝数、桃数和籽棉产量方面均显著低于对照CCRI24和24-epi-BL处理pag1。6)为了进一步揭示BRs调控棉花耐旱性的作用机制,我们采用i TRAQ方法检测6%PEG6000处理0 h,12 h,24 h,36 h条件下的pag1和CCRI24中的差异蛋白,在pag1/CCRI24±1.2(P value0.05)的筛选条件下共鉴定到2100个差异蛋白,其中上调蛋白数目为1132个,下调蛋白数目为1105个,上调和下调共同拥有蛋白数目为137个。GO(Gene ontology)分析显示上调蛋白主要聚类于细胞进程、代谢过程和刺激响应等12类中,下调表达的差异蛋白主要聚类于刺激响应、大分子复合物和代谢过程等13类中。在下调表达的差异蛋白中,有许多是参与干旱胁迫响应的,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缺失突变体pag1,从根系、气孔、能量合成、基因表达和蛋白组学等方面全面揭示了BRs缺失造成植物对干旱胁迫敏感的作用机制,使我们认识了BRs在生长发育和干旱胁迫中的重要作用,为我们如何利用植物激素BRs和为我们利用分子生物学方法克隆BRs相关基因以培育抗旱性强、高产稳产的棉花新品种提供了重要的理论依据。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S562

【参考文献】