枳NLP转录因子克隆及其在不同水分条件下的表达

摘要

Objective]The objective of this study was to analyze the expression ofPoncirus trifoliata (L.) Raf. NIN-like transcription factors to discuss the regulation mechanism of nitrogen assimilation under different water conditions in citrus.[Method] Primers were designed to amplify the sequences of theNLPgenes CDS ofPoncirus trifoliate(L.) Raf.based on orange genome databases and molecular biology. Alignment of the sequences was performed using ClustalX and phylogenetic analysis of this alignment was conducted using MEGA. Analysis of the RelativeNLP genes expression data under different water conditions using Real-Time Quantitative PCR.[Result]FourPoncirus trifoliata (L.) Raf.NLP genes:PtNLP2,PtNLP4,PtNLP7andPtNLP8were obtained. The sequence alignment analysis showed that the identity of all NIN-like proteins was 45.13%, and possessed both the RWP-RK and PB1 domain, and all NIN-like proteins were high identity to sweet orange, respectively 97.57%, 96.47%, 99% and 97.33%. Phylogenic analysis has indicated that the 4Poncirus trifoliata (L.) Raf.NLP genes can be classified into four groups with Arabidopsis (PtNLP2andAtNLP1/2,PtNLP4andAtNLP4/5,PtNLP7andAtNLP6/7,PtNLP8andAtNLP8/9). There were differences in the expression pattern ofNLP genes inPoncirus trifoliata (L.) Raf. leaves and roots. The results indicated that with the decrease of soil water, the expression level ofNLP genes inPoncirus trifoliata (L.) Raf. leaves were up-regulated. ThePtNLP2, PtNLP4,PtNLP7 andPtNLP8 expression level were the highest when the relative water capacity was 15.4%, they were 2.9, 3.5, 5.9 and 2.8 folds higher respectively compared to the control (the relative water holding capacity was 61.0%). After that, the gene expression level began to down-regulate and had no statistically significant difference between the control and the relative water holding capacity of 9.4%. While the expression level ofNLP genes inPoncirus trifoliata (L.) Raf. roots of control were the highest, and the gene expression pattern showed a down-regulated trend accompanied by the loss of soil water and the difference was statistically significant. ThePtNLP2,PtNLP4,PtNLP7 andPtNLP8 expression level of roots were maximum down-regulated by 6.7, 2.8, 4.8 and 2.3 folds respectively compared with the control. The expression ofNLP genes inPoncirus trifoliata (L.) Raf. leaves and roots remained at the low expression level after rewatering and had statistically significant difference compared with the control.[Conclusion]The expression of Poncirus trifoliata (L.) Raf.NLP genes is closely related to the water condition of soil. The expression level ofNLP genes inPoncirus trifoliata (L.) Raf. leaves were up-regulated by drought stress, and then down-regulated when the water deficit was very serious. But the expression level ofNLP genes inPoncirus trifoliata (L.) Raf. roots were down-regulated continuously accompanied by the loss of soil water, and expression of thePtNLP2 andPtNLP7 had a great change in roots.%【目的】分析柑橘砧木枳（Poncirus trifoliata (L.) Raf.）参与氮素吸收与同化的重要转录因子NLP在干旱胁迫下的表达模式，为柑橘在干旱胁迫条件下对氮素吸收与同化的调控机理提供研究参考。【方法】以柑橘砧木枳为试验材料，在甜橙（Citrus sinensis (L.) Osb.）基因组数据库的基础上，利用生物信息学筛选获得甜橙NLP，并以此设计引物扩增枳NLP的CDS全长并测序。利用ClustalX和MEGA程序进行序列比对和系统发育分析，利用实时荧光定量PCR技术分析不同水分条件下的枳NLP的表达模式。【结果】筛选并克隆获得了4条枳NLP序列，为PtNLP2、PtNLP4、PtNLP7和PtNLP8。序列比对分析表明枳NLP之间的蛋白质序列一致性为45.13%，均具有明显的RWP-RK和PB1结构域。枳与甜橙的NLP蛋白质序列的一致性极高，分别为97.57%、96.47%、99.0%及97.33%。系统发育分析表明枳NLP可分为4个进化类型，与拟南芥的NLP分类一致，即PtNLP2与AtNLP1/2一类， PtNLP4与AtNLP4/5一类，PtNLP7与AtNLP6/7一类，PtNLP8与AtNLP8/9一类。在不同水分条件下，枳NLP的表达模式在叶与根中存在差异。随着基质水分含量的减少，枳叶NLP呈现先上升再下降的趋势。与对照（相对持水量为61.0%）相比，叶PtNLP2、PtNLP4、PtNLP7及PtNLP8的表达量在相对持水量为15.4%时上调至最高水平，分别上调了2.9、3.5、5.9及2.8倍，之后持续下调；到相对持水量为9.4%时，其表达水平低于对照但无显著差异。而枳根 NLP的表达量在对照中最高，后随着水分的散失呈总体下降趋势且差异显著，与对照相比，根 PtNLP2、PtNLP4、PtNLP7及PtNLP8的最大下调倍数分别为6.7、2.8、4.8及2.3。复水后，枳叶与根NLP的表达量低于复水前，且与对照差异显著。【结论】枳NLP的表达与土壤的水分条件密切相关。枳叶片NLP的表达水平在干旱胁迫前、中期上调，后期下调；而枳根NLP的表达水平在干旱胁迫下持续下调。其中，PtNLP2和PtNLP7的表达量在枳根响应干旱胁迫中变化较大。