青杄转录因子PwNAC30及其启动子序列的克隆与表达分析

Cloning and Analysis of a Transcription Factor PwNAC30 and the Promoter Sequence in Picea wilsonii

该研究以青杄(Picea wilsonii)为实验材料,通过PCR从青杄的cDNA文库中克隆得到一个NAC转录因子,命名为PwNAC30。生物信息学分析显示,PwNAC30开放阅读框1 179bp,共编码392个氨基酸,在其N端存在保守的NAM(no apical meristem)结构域,可分为A～E等5个亚结构域。多序列对比和系统进化树分析显示,PwNAC30蛋白与同为云杉属的北美云杉(Picea sitchensis)聚为一类。启动子克隆分析显示,PwNAC30基因启动子上存在脱落酸(ABA)、赤霉素(GA)、茉莉酸甲酯(MeJA)、TC-rich repeats等激素和逆境响应元件,在GA、ABA、MeJA、低温、干旱、盐的处理下,其启动子活性均明显增强。荧光定量PCR分析表明,PwNAC30在球果中的表达量最高,而在花粉和种子中的表达量最低。PwNAC30对于盐、干旱、低温、ABA、MeJA、GA处理均有响应,尤其对盐、干旱、MeJA的响应最为显著。亚细胞定位结果显示,PwNAC30蛋白定位于细胞核与细胞质,主要定位于细胞核中。酵母单杂及双杂结果表明,PwNAC30蛋白的全长和N端没有转录激活活性,而C端有转录激活活性,且PwNAC30自身能形成同源二聚体。研究表明,青杄PwNAC30基因可以作为一个转录因子发挥作用,其转录激活活性在C端,且自身能够形成同源二聚体结构;PwNAC30基因广泛参与了ABA、GA、MeJA等激素的信号通路,并对盐、干旱、低温处理有响应。

In this study,Picea wilsonii was used as experimental material to clone a NAC transcription factor from cDNA library by PCR,which was named PwNAC 30.Bioinformatics analysis showed that PwNAC 30 open reading frame was 1 179 bp,encoding a total of 392 amino acids,a conserved NAM (no apical meristem) domain at its N-terminus and can be divided into five sub-domains of A-E.Multi-sequence comparison and phylogenetic tree analysis showed that PwNAC30 protein was clustered with North American spruce ( P.sitchensis ) of the Picea .Promoter cloning analysis showed that there were hormones and stress response elements such as gibberellin (GA),abscisic acid (ABA),methyl jasmonate (MeJA),TC-rich repeats on the PwNAC 30 promoter.Its promoter activity was significantly enhanced under ABA,GA,MeJA,low temperature,drought and salt treatments.Real-time PCR analysis showed that PwNAC 30 had the highest expression in cones and the lowest in pollens and seeds.PwNAC 30 responded to salt,drought,low temperature,ABA,MeJA,GA treatments,especially for salt,drought and MeJA.Subcellular localization experiments showed that PwNAC30 protein is localized in the nucleus and cytoplasm,mainly in the nucleus.Yeast one hybrid and two hybrid experiments showed that PwNAC30 protein had no transcriptional activation activity at its full length and N-terminus,whereas its C-terminus had transcriptional activation activity.PwNAC30 can form homodimers by itself.Studies have shown that PwNAC 30 is a transcription factor with transcriptional activation activity at the C-terminus and is capable of forming a homodimeric structure by itself.It is widely involved in the signaling pathways of ABA,GA,MeJA and other hormones,and responds to salt,drought and low temperature.