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  

Isolation of Chlorophyll Metabolism Key Genes and Molecular Mechanism of Green Fade in Guzmania Bracts Discoloration Process

作　　者：刘建新 丁华侨 田丹青 王炜勇 刘慧春 

机构地区：[1]浙江省萧山棉麻研究所,杭州311202

出　　处：《中国农业科学》2016年第13期2593-2602,共10页Scientia Agricultura Sinica

基　　金：浙江省自然科学基金(LQ13C150002);浙江省公益性技术应用研究计划(2012C22085)

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c DNA文库构建及EST批量测序获得叶绿素合成关键酶:谷氨酰t RNA合成酶基因(glutamyl-t RNA 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(Gen Bank:KP144289),序列长为1 140 bp,编译171AA的蛋白氨基酸序列,并存在Gln RS-cataytic core和Nt-trans superfamily保守区;获得的UROS(Gen Bank:KP144288)c DNA序列长为613 bp,编译188AA的蛋白氨基酸序列,并存在Hem D和Hem D Superfamily保守区;获得的PPH(Gen Bank:KP723523)c DNA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[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 eDNA 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 enco

关 键 词：擎天凤梨 叶绿素 谷氨酰t RNA合成酶基因 尿卟啉原-Ⅲ合酶基因 脱镁叶绿素酶 

分 类 号：S682.36[农业科学—观赏园艺]