拟南芥光呼吸突变体pr1的基因克隆及其生理特性研究

发布时间：2018-04-11 08:55

本文选题：拟南芥 + 光呼吸　；参考：《华南农业大学》2016年硕士论文

【摘要】：光呼吸是C3植物中仅次于光合作用的第二大代谢流,正常条件下C3植物的光呼吸可损失光合产物的20-30%。毫无疑问,光呼吸限制了植物的光能利用效率,最终影响作物产量。然而,大量的研究表明,清除或调节光呼吸代谢途径,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,这表明光呼吸不仅仅只与光合相关,对植物的生存也有重要的生物学意义。目前已知光呼吸与氮的同化、呼吸作用、氨基酸代谢、一碳代谢和氧化还原信号等代谢过程密切相关。通过筛选光呼吸突变体,挖掘光呼吸代谢调控新基因,将有助于进一步解析光呼吸与其他代谢途径之间的相互调节关系。本研究以模式植物拟南芥的Columbia(Col-0)生态型为实验材料,通过EMS诱变构建突变体库,利用光呼吸突变体在正常大气条件下出现黄化、矮小或致死,而在高CO2条件下正常生长的表型进行光呼吸突变体筛选。筛选出突变体命名为pr1(Photorespiration related 1),对突变体进行了生理、分子和遗传学的分析,研究结果如下:1.表型生理分析:在正常大气条件下,突变体pr1同野生型(Col-0)相比植株矮小,叶片颜色发黄,而在高CO2条件下两者差别较小。正常大气条件下突变体pr1甘氨酸含量显著高于Col-0。2.基因定位:突变体pr1与Landsberg erecta(Ler)生态型拟南芥杂交,利用F2代植株进行图位克隆,确定其基因定位区间位于第4条染色体17.97 Mb附近。通过对pr1全基因组测序分析,确定其突变基因座位号为AT4G38380,编码多药和有毒化合物排出家族蛋白(Multidrug and toxic compound extrusion,MATE),本研究中暂命名为MATE1蛋白。3.互补实验:构建载体proMATE1::MATE1-Flag转化pr1,转化株系在大气生长条件下,叶片颜色、植株形态等均恢复正常。4.组织表达:构建载体proMATE1::GUS转化Col-0,对转化株系GUS染色分析表明MATE1主要在新叶、茎分生组织、根、花药、果荚中。Real-time PCR检测发现强光处理4h后MATE1表达含量急剧上升。5.亚细胞定位:构建35S::MATE1-GFP转化Col-0原生质体,对GFP荧光分析将MATE1定位于叶绿体膜上。6.筛选mate1的T-DNA插入突变体(CS300818),命名为mate1-1,mate1-1植株具有矮小、叶片卷曲,基本不结实的表型。7.构建35S::MATE1转化Col-0,转化株系与Col-0相比其生物量明显增加。综上所述,本研究工作筛选了光呼吸突变体pr1,图位克隆、全基因组测序分析和基因互补实验表明突变基因为MATE1,MATE1定位在叶绿体膜上,其T-DNA突变体mate1-1有明显的发育缺陷,过量表达MATE1增加了生物量。MATE1影响光呼吸的作用机理仍有待进一步研究。
[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.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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