發(fā)布時間：2018-04-11 08:55

  本文選題：擬南芥 + 光呼吸��； 參考：《華南農業(yè)大學》2016年碩士論文

【摘要】：光呼吸是C3植物中僅次于光合作用的第二大代謝流,正常條件下C3植物的光呼吸可損失光合產物的20-30%。毫無疑問,光呼吸限制了植物的光能利用效率,最終影響作物產量。然而,大量的研究表明,清除或調節(jié)光呼吸代謝途徑,均未能有效提高植物的光合作用,反而造成光合速率下降或者致死,這表明光呼吸不僅僅只與光合相關,對植物的生存也有重要的生物學意義。目前已知光呼吸與氮的同化、呼吸作用、氨基酸代謝、一碳代謝和氧化還原信號等代謝過程密切相關。通過篩選光呼吸突變體,挖掘光呼吸代謝調控新基因,將有助于進一步解析光呼吸與其他代謝途徑之間的相互調節(jié)關系。本研究以模式植物擬南芥的Columbia(Col-0)生態(tài)型為實驗材料,通過EMS誘變構建突變體庫,利用光呼吸突變體在正常大氣條件下出現黃化、矮小或致死,而在高CO2條件下正常生長的表型進行光呼吸突變體篩選。篩選出突變體命名為pr1(Photorespiration related 1),對突變體進行了生理、分子和遺傳學的分析,研究結果如下:1.表型生理分析:在正常大氣條件下,突變體pr1同野生型(Col-0)相比植株矮小,葉片顏色發(fā)黃,而在高CO2條件下兩者差別較小。正常大氣條件下突變體pr1甘氨酸含量顯著高于Col-0。2.基因定位:突變體pr1與Landsberg erecta(Ler)生態(tài)型擬南芥雜交,利用F2代植株進行圖位克隆,確定其基因定位區(qū)間位于第4條染色體17.97 Mb附近。通過對pr1全基因組測序分析,確定其突變基因座位號為AT4G38380,編碼多藥和有毒化合物排出家族蛋白(Multidrug and toxic compound extrusion,MATE),本研究中暫命名為MATE1蛋白。3.互補實驗:構建載體proMATE1::MATE1-Flag轉化pr1,轉化株系在大氣生長條件下,葉片顏色、植株形態(tài)等均恢復正常。4.組織表達:構建載體proMATE1::GUS轉化Col-0,對轉化株系GUS染色分析表明MATE1主要在新葉、莖分生組織、根、花藥、果莢中。Real-time PCR檢測發(fā)現強光處理4h后MATE1表達含量急劇上升。5.亞細胞定位:構建35S::MATE1-GFP轉化Col-0原生質體,對GFP熒光分析將MATE1定位于葉綠體膜上。6.篩選mate1的T-DNA插入突變體(CS300818),命名為mate1-1,mate1-1植株具有矮小、葉片卷曲,基本不結實的表型。7.構建35S::MATE1轉化Col-0,轉化株系與Col-0相比其生物量明顯增加。綜上所述,本研究工作篩選了光呼吸突變體pr1,圖位克隆、全基因組測序分析和基因互補實驗表明突變基因為MATE1,MATE1定位在葉綠體膜上,其T-DNA突變體mate1-1有明顯的發(fā)育缺陷,過量表達MATE1增加了生物量。MATE1影響光呼吸的作用機理仍有待進一步研究。
[Abstract]:Photorespiration is the second largest metabolic flow in C3 plants after photosynthesis. Under normal conditions, photorespiration of C3 plants can lose 20-30% of photosynthetic products.There is no doubt that light respiration limits the efficiency of light use in plants and ultimately affects crop yields.However, a large number of studies have shown that the removal or regulation of photorespiration pathways can not effectively improve the photosynthesis of plants, but lead to a decrease in photosynthetic rate or death, which indicates that photorespiration is not only related to photosynthesis.It also has important biological significance for the survival of plants.At present, photorespiration is closely related to nitrogen assimilation, respiration, amino acid metabolism, carbon metabolism and redox signal.The screening of photorespiratory mutants and the discovery of new genes for regulation of photorespiration metabolism will help to further analyze the interregulatory relationship between photorespiration and other metabolic pathways.In this study, the model plant of Arabidopsis thaliana Col-0) was used as the experimental material. The mutant library was constructed by EMS mutation. The mutant was yellow, dwarf or lethal under normal atmospheric conditions.The normal growth phenotypes under high CO2 conditions were screened for photorespiratory mutants.The mutant, named pr1(Photorespiration related 1, was selected for physiological, molecular and genetic analysis. The results are as follows: 1.Phenotypic physiological analysis: compared with wild type Col-0, pr1 mutant was smaller and yellowish in leaf color under normal atmospheric conditions, but the difference was small under high CO2 condition.The pr1 glycine content of the mutant was significantly higher than that of Col-0.2 under normal atmospheric conditions.Gene location: pr1 and Landsberg erecta er) ecotypic Arabidopsis thaliana were hybridized. Using F2 generation plants for mapping cloning, it was determined that the region of gene location was about 17.97Mb on chromosome 4.By sequencing and analyzing the whole genome of pr1, the mutant gene was identified as AT4G38380, encoding multidrug and toxic compound extrusionmate, which was tentatively named MATE1 protein.Complementary experiment: the vector proMATE1::MATE1-Flag was constructed to transform PR1. Under the condition of atmospheric growth, the leaf color and plant morphology of the transformed strain returned to normal. 4.Tissue expression: construction vector proMATE1::GUS was transformed into Col-0. The results of GUS staining showed that MATE1 was mainly detected in leaves, stem meristem, roots, anthers and pods. Real-time PCR detection showed that MATE1 expression increased sharply 4 hours after intense light treatment.Subcellular localization: 35S::MATE1-GFP was constructed to transform Col-0 protoplasts, and MATE1 was located on chloroplast membrane by GFP fluorescence analysis.The T-DNA insertion mutant CS300818 of mate1, named mate1-1, has a dwarf, curly leaf, and basically sterile phenotype.When 35S::MATE1 was constructed to transform Col-0, the biomass of transformed strain was significantly higher than that of Col-0.In conclusion, the photorespiratory mutants PR1, map-based cloning, genome sequencing and gene complementation experiments showed that the mutant T-DNA mutant mate1-1 had obvious developmental defects because it was located on chloroplast membrane.The mechanism of overexpression of MATE1 to increase the biomass. MATE1 effect on photorespiration remains to be further studied.
【學位授予單位】：華南農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2

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