擎天凤梨苞片叶绿素代谢关键基因的分离及褪绿的分子机理

doi:10.3864/j.issn.0578-1752.2016.13.015

摘要/Abstract

摘要： 【目的】擎天凤梨是一种新潮花卉，其苞片在成花变色过程中常常伴随着绿色的逐渐消退。分离叶绿素生物合成与降解途径的关键基因，探索在苞片变色过程中叶绿素代谢的分子基础和机理，有助于解析呈色苞片中绿色退化的原因。【方法】通过cDNA文库构建及EST批量测序获得叶绿素合成关键酶：谷氨酰tRNA合成酶基因（glutamyl-tRNA synthetase，GTS）和尿卟啉原-Ⅲ合酶基因（uroporphyrinogen-Ⅲsynthase，UROS）；通过同源克隆技术获得叶绿素降解关键酶：脱镁叶绿素酶（pheophytin pheophorbide hydrolase/pheophytinase，PPH）基因；通过采用透光系数法测量叶绿素含量、HPLC法测定类黄酮含量以及采用实时定量PCR法测定叶绿素代谢关键基因的基因表达模式，探索呈色苞片中绿色部分消失的分子机理。【结果】获得的GTS（GenBank：KP144289），序列长为1 140 bp，编译171AA的蛋白氨基酸序列，并存在GlnRS-cataytic core 和Nt-trans superfamily保守区；获得的UROS（GenBank：KP144288）cDNA序列长为613 bp，编译188AA的蛋白氨基酸序列，并存在HemD和HemD Superfamily保守区；获得的PPH（GenBank：KP723523）cDNA序列长为266 bp，编译88AA的蛋白氨基酸序列，并存在Abhydrolase-6保守区。通过对不同变色阶段苞片的叶绿素、类黄酮含量测定以及叶绿素代谢相关基因的表达水平分析，可知苞片变色过程伴随着叶绿素含量的显著降低和类黄酮含量的明显增加，同时叶绿素合成相关的GTS和UROS表达水平也明显降低，而叶绿素降解关键酶基因PPH的表达水平在苞片变色初期显著上升，在变色完成后则降低到接近绿叶状态的最低水平。而作为对照材料的绿叶，其叶绿素含量是最高的，而类黄酮含量是最低的，同时叶绿素的合成相关酶基因表达量最高，而降解相关酶基因表达量最低，这与彩叶植物中叶绿素和类黄酮色素的表现较为一致。【结论】获得了擎天凤梨的叶绿素合成相关酶基因GTS和UROS以及降解关键酶基因PPH。由于叶绿素合成量减少，降解量显著增加引起叶绿素含量的降低，从而影响苞片变色过程中绿色的消退，并伴随类黄酮含量的增加。PPH在叶绿素的降解过程中起关键性作用。

关键词: 擎天凤梨, 叶绿素, 谷氨酰tRNA合成酶基因, 尿卟啉原-Ⅲ合酶基因, 脱镁叶绿素酶

Abstract: 【Objective】 Guzmania is a trendy flower. Its flower formation and bracts discoloration process are often accompanied by green fade. To analyze the reason which bract’s green fade, it is necessary to isolate key genes and explore molecular mechanism of chlorophyll biosynthesis and degradation metabolism in the process of bracts discoloration. 【Method】 By cDNA library construction and EST sequencing batch, two key enzyme genes in chlorophyll biosynthesis pathway, including glutamyl-tRNA synthetase (GTS) and Uroporphyrinogen-Ⅲ synthase (UROS), were obtained. By homology cloning technology, a key enzyme gene in chlorophyll degradation pathway, pheophytin pheophorbide hydrolase gene (PPH), was obtained. Through measuring chlorophyll content using light transmittance measurement method, flavonoid content using HPLC method and analyzing gene expression pattern of key genes in chlorophyll metabolism using real-time quantitative PCR method, the molecular mechanism which green fade in discoloration bracts was studied. 【Result】 GTS obtained has 1 140 bp (GenBank: KP144289) in cDNA sequence, which compiles a 171 amino acid protein sequence, and has GlnRS-cataytic core and Nt-trans superfamily conservative regions; UROS obtained has 613 bp (GenBank: KP144288) in cDNA sequence, which compiles a 188 amino acid protein sequence, and has HemD and HemD Superfamily conservative regions; PPH obtained has 266 bp (GenBank: KP723523) in cDNA sequence, which compiles a 88 amino acid protein sequence, and has Abhydrolase-6 conservative region. Through measuring chlorophyll, flavonoid content and studying the expression level of key genes in chlorophyll metabolism, it could know that bract discoloration process accompanied by significant reduction of chlorophyll content and significant increase of flavonoid content. Furthermore, the expression level of chlorophyll biosynthesis key enzyme’s encoding genes, GTS and UROS, were also decreased obviously. As for the chlorophyll degradation key enzyme’s encoding gene, PPH, its expression level increased significantly at the beginning of bracts discoloration, then reduced to close to the lowest level, which in accord with green leaves, after finishing of discoloration. As for the control material, green leaf had the highest chlorophyll content and the lowest flavonoid content. Furthermore, the expression amount of chlorophyll biosynthesis related genes were the highest, while that of degrade related genes was the lowest. This was consistent with colorful plants in chlorophyll and flavonoid pigment change trend. 【Conclusion】 We obtained biosynthesis key enzymes genes, GTS, UROS, and degradation key enzyme gene, PPH, in chlorophyll metabolism pathway from Guzmania. The reason that bracts’ green fade was due to reduction of chlorophyll content, and accompanied by increase of flavonoid content. The reduction of chlorophyll content was due to chlorophyll’s biosynthesis reduced and degradation increased significantly. PPH played a key role in chlorophyll degradation process. The results will provide a basis for researching pigment change of view bract plants.

Key words: Guzmania Ruiz&Pav, chlorophyll, glutamyl-tRNA synthetase, uroporphyrinogen-III synthase, pheophytin pheophorbide hydrolase/pheophytinase

刘建新，丁华侨，田丹青，王炜勇，刘慧春. 擎天凤梨苞片叶绿素代谢关键基因的分离及褪绿的分子机理[J]. 中国农业科学, 2016, 49(13): 2593-2602.

LIU Jian-xin, DING Hua-qiao, TIAN Dan-qing, WANG Wei-yong, LIU Hui-chun. Isolation of Chlorophyll Metabolism Key Genes and Molecular Mechanism of Green Fade in Guzmania Bracts Discoloration Process[J]. Scientia Agricultura Sinica, 2016, 49(13): 2593-2602.

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