U+3B8FNPR1同源基因全长cDNA的分离与表达分析

 NPR1是水杨酸诱导抗病基因表达的重要调控因子之一，主要作用于水杨酸信号转导途径的上、下游。采用稀释池PCR法，筛选U+3B8F（Prunus salicina var. cordata）叶片全长均一化cDNA文库，分离U+3B8F叶片NPR1同源基因的全长cDNA；用不同浓度的水杨酸喷施一年生U+3B8F嫁接苗嫩叶，采用荧光定量PCR技术检测所获得的NPR1同源基因的表达量差异。试验结果表明，共获得了4个NPR1同源基因的全长cDNA，分别命名为PsNPR1、PsNPR3.1、PsNPR3.2和PsNPR3.3，其长度分别为2 442、1 827、2 244和2 615 bp，其ORF长度分别为1 788、1 770、1 767 和1 782 bp，分别编码596、590、589和594个氨基酸的蛋白质；氨基酸序列比对结果表明，U+3B8F这4个NPR1同源基因的编码区均含有BTB/POZ和ANK两个保守域及核定位信号（NLS）；PsNPR1与拟南芥的AtNPR1和AtNPR2聚为一簇，PsNPR3.1、PsNPR3.2和PsNPR3.3与拟南芥的AtNPR3和AtNPR4聚为另一簇。PsNPR1在喷施1.0 mmol ? L-1水杨酸的叶片中表达量最高；PsNPR1、PsNPR3.1、PsNPR3.2在响应1.0 mmol .  L-1水杨酸诱导过程中表达量持续上升， 30 h表达量最高，之后呈下降趋势，因此，水杨酸诱导抗病基因高表达的最佳处理浓度为1.0 mmol ? L-1，最佳响应时间为30 h。

Isolation and Expression Analysis of Full-length cDNAs of NPR1 Homologous Genes in Nai（Prunus salicina var. cordata）

NPR1 is one of important regulatory factors inducing expression of desease-resistant genes，and mainly acts in the up- or down-streams on salicylic acid signal transduction pathway. Full-length cDNAs of NPR1 homologous genes were screened from enriched full-length normalized cDNA library by pool-dilued PCR method in Nai’s leaves；young leaves in one-year grafted seedlings were sprayed with salicylic acid solutions of different concentrations，the differences in expression levels of the isolated NPR1 genes were detected by fluorescence quantitative PCR. The results showed that 4 full-length cDNAs of NPR1 homologous genes were obtained and named as PsNPR1，PsNPR3.1，PsNPR3.2 and PsNPR3.3 respectively；their responding full lengths were 2 442，1 827，2 244 and 2 615 bp；their reponding lengths of ORFs were 1 788，1 770，1 767 and 1 782 bp，encoding proteins containing 596，590，589 and 594 amino acids，respectively；the results of amino acid sequence alignment showed that coding regions of the 4 NPR1 homologous genes contained two conservative domains（BTB/POZ and ANK）and nuclear localization signal（NLS）；Nai’s PsNPR1 was clustered with Arabidopsis thaliana’s AtNPR1 and AtNPR2 forming a cluster，Nai’s PsNPR3.1，PsNPR3.2 and PsNPR3.3 were clustered with Arabidopsis thaliana’s AtNPR3 and AtNPR4 forming another cluster. The expression level of PsNPR1 was the highest in Nai’s young leaves sprayed with 1.0 mmol ? L-1 salicylic acid solutions；the expression levels of PsNPR1，PsNPR3.1 and PsNPR3.2 continuously increased in process of response to 1.0 mmol ? L-1 salicylic acid，and reached maximum at 30 hours after spraying，then tended to decrease，therefore，the optimal spraying concentration of salicylic acid was 1.0 mmol ? L-1，the optimal responsing time of 1.0 mmol ? L-1 salicylic acid was 30 hours.