日本结缕草ZjNAC3基因的克隆与功能分析

Cloning,Expression Analysis,Subcellular Localization and Transcriptional Activation Activity Characteristics of ZjNAC3 Gene of Zoysia japonica

从日本结缕草中克隆获得ZjNAC3基因,编码区全长1167bp,并获得含1622bp的启动子序列。保守结构域分析表明,克隆的ZjNAC3基因属于NAC转录因子家族。启动子分析表明,启动子中含有多个激素、胁迫响应元件。脱落酸(ABA)、乙烯(ETH)、茉莉酸甲酯(MeJA)、氯化钠(NaCl)、甘露醇(Mannitol)均能够负调控ZjNAC3启动子活性,水杨酸(SA)能够正调控ZjNAC3启动子活性。ZjNAC3在日本结缕草根、茎、叶中均有表达,叶中表达量较高,且衰老叶片中的表达水平显著高于其他时期。激素和胁迫处理后,ZjNAC3表达水平受ABA、MeJA、ETH和干旱、高盐胁迫的调控。亚细胞定位结果显示,ZjNAC3定位于细胞核中。转录自激活验证表明,ZjNAC3具有转录自激活活性,进一步分析发现其N端不具有转录激活活性,其C端具有转录激活活性。

The ZjNAC3 gene with a full-length coding region of 1167bp was cloned from Zoysia japonica,and promoter sequence containing 1622bp was obtained through PCR amplification.Conserved domain analysis indicated that the cloned ZjNAC3 gene belonged to the NAC transcription factor family.Cis-elements analysis of promoter indicated that the promoter contained multiple hormones and stresses response elements.GUS staining results showed abscisic acid(ABA),ethylene(ETH),methyl jasmonate(MeJA),sodium chloride(NaCl)and mannitol could negatively regulate the activity of ZjNAC3 promoter,while salicylic acid(SA)could positively regulate the activity of ZjNAC3 promoter.ZjNAC3 was expressed in roots,stems,and leaves of Z.japonica,and the expression level in leaves was higher,and the expression level in senescent leaves was significantly higher than that in other periods.After hormone and stress treatment,the expression level of ZjNAC3 was regulated by ABA,MeJA,ETH,drought or high salt stress.Subcellular localization showed that ZjNAC3 was located in the nucleus.The verification of transcriptional self-activation showed that ZjNAC3 had transcriptional self-activation activity.Further analysis showed that its the C-terminus,but not the N-terminus,was responsible for the transcriptional activation activity.