柱花草9-顺式环氧类胡萝卜素双加氧酶基因(SgNCED1)的克隆及表达分析

Cloning and characteristics of 9-cis-epoxycarotenoid dioxygenase gene (SgNCED1) from Stylosanthes guianensis

9-顺式环氧类胡萝卜素双加氧酶（NCED）是高等植物ABA生物合成途径中的关键酶，本研究以3种豆科植物NCED的同源序列设计简并引物，用RT—PCR结合RACE以及嵌套PCR的方法。从柱花草叶片中克隆了一个编码NCED的基因，命名为SgNCED1。其cDNA全长2241bp，开放阅读框架为1809bp，编码602个氨基酸。Southern杂交结果表明，该基因在柱花草基因组中以单拷贝形式存在。氨基酸序列同源比对和系统进化分析表明SgNCED1与花生的AhNCED1高度同源（92％），与另外3种豆科植物的NCED也有很高的同源性（72％～75％）。亚细胞定位预测表明SgNCED1蛋白的N-末端具有叶绿体转运肽。叶片脱水诱导的SgNCED1基因的表达与内源ABA的积累、变化同步。

9-cis-epoxycarotenoid dioxygenase （NCED） is a key enzyme for ABA biosynthesis in higher plants. Degenerate primers were synthesized based on highly homologous regions from the NCED sequences of three leguminous plants and were used for cloning an NCED gene from dehydrated leaves of S. guianensis using RT- PCR, RACE, and nesting PCR. A full length of NCED cDNA, namely SgNCED1, was cloned and sequenced. It was found to be 2 241 bp long and contained an open reading frame （ORF） of 1 809 bp which encodes a peptide of 602 amino acids. Southern blot analysis showed that SgNCED1 was a single copy gene in the genome of S. guianensis. Multiple sequence alignments of the deduced amino sequences showed that the SgNCED1 protein shared 92% identity with AhNCED1 from Arachis hypogaea and was highly homologous （72%-75%） to other NCED of three leguminous plants as well. A chloroplast transit peptide located at the N-terminus of the SgNCED1 was predicted by iPSORT algorithm. Dehydration induced the expression of SgNCED1 and ABA accumulation in S. guianensis.