无核荔枝ABA生物合成关键酶LcNCED基因克隆及其在生理落果阶段中的表达分析

9-顺-环氧类胡萝卜素双加氧酶(9-cis-epoxycarotenoids dioxygenase,NCED)是ABA生物合成途径的关键限速酶,本研究利用RT-PCR技术从A4无核荔枝中分离得到NCED的3条c DNA序列(命名为LcNCED1、LcNCED2和LcNCED3)。通过同源比对和系统进化树分析,LcNCED基因编码的氨基酸序列与拟南芥等30个物种中的NCED氨基酸序列同源性都在65%以上,从而确定LcNCED属于NCED家族;通过与7条拟南芥及5条苹果NCED家族基因进行系统进化树分析,LcNCED2与调控苹果花瓣脱落MdNCED2、MdNCED4基因具有较高的同源性。Real-Time PCR表达分析发现,3个LcNCED基因在不同样品间表达存在显著差异,其中LcNCED2在各样品间的表达水平显著高于LcNCED1和LcNCED3,且LcNCED2表现出显著的组织特异性表达特点,花后15~75 d,LcNCED2在离区(果柄离层部位上下0.2 cm)中的表达量均高于种脐和果皮中,在花后45 d达到峰值,花后75 d稍有下降,基因表达的峰值略早于A4无核荔枝落果高峰期。而LcNCED1和LcNCED3在各组织样品中基因表达变化趋势规律不明显。上述结果表明,LcNCED2在离区中表达量变化比较符合A4无核荔枝生理落果趋势,推断LcNCED2基因可能与无核荔枝采前落果关系密切。 

Expression Analysis and Cloning of NCED Gene During Physiological Fruit Drop Period of Seedless Litchi Fruit

9-cis-epoxycarotenoids dioxygenase （NCED） is the key enzyme for ABA synthesis in indirect pathway. Three full-length cDNA（temporarily named as LcNCED1, LcNCED2 and LcNCED3） encoding 9-cis-epoxycarotenoids dioxygenase were obtained from the litchi genome sequence resource （unpublished）, and were isolated with the techniques of RT-PCR. Phylogenetic tree analysis showed that amino acids sequence of LcNCED genes shared more than 65% identity with the sequence of NCED from other 30 plants； Compared with 7 NCED family gene in Arabidopsis thaliana and 5 NCED family gene in Malus domestica, which showed high homology with AtNCED genes, LcNCED2 had the highest amino acids sequence identity with MdNCED2 and MdNCED4 which can control petals abscission. Real-time PCR results showed that LcNCED2 expression level was significantly higher than that of LcNCED1 and LcNCED3 in each sample. LcNCED2 showed significant characteristics of tissue specific expression,   the expression in abscission zone presented higher than that in hilum and pericarp. The expression level of LcNCED2 increased gradually during fruit development, and a rapid increase occurred  in  45  DAF  stages  which  is  a  little earlier than the fruit drop peak stage, The trend of expression of LcNCED2 in abscission zone was consistent with  the trend of seedless litchi physiological fruit drop, the trend of expression of LcNCED1 and LcNCED3 were irregular. Thus, we considered that LcNCED2 gene might be the key gene invovled in fruitlet abscission in litchi.