Functional Analysis Of The NAC Transcription Factor Genes AmNAC2 And AmNAC4 From Ammopiptanthus Mongolicus

The NAC（NAM,ATAF and CUC）transcription factors are closely related to stress tolerance as well as growth and development of plants.Ammopiptanthus is the unique evergreen broadleaf plant endemic to the desert regions of Northwest China,which has very high stress tolerance and consists of two species named A.mongolicus and A.nanus.In this study,the transcriptional activation activity of the protein encoded by AmNAC2 from A.mongolicus was analyzed by using a yeast self-activation system,and the expression analysis of this gene was conducted by RT-q PCR.Moreover,both transgenic poplar（Shan xin）and transgenic rice（Zhonghua11）lines with the AmNAC4 gene from this species were constructed,and salt tolerance of the transgenic poplar lines was evaluated.The main results are as follows:1.Both the full-length AmNAC2 protein（of 292 amino acids）and its C-terminal region（amino acids 159-292）have transcriptional activation activity,while its N-terminal region（amino acids 1-158）has not.2.In laboratory-cultured A.mongolicus seedlings,the expression level of AmNAC2 increased rapidly and significantly under low temperature,salt and high temperature stress treatments,but it was up-regulated slowly under drought treatment.In young leaves of naturally growing A.mongolicus plants in the wild,the expression levels of AmNAC2 were obviously higher in autumn and winter than in spring and summer.During the spring period,AmNAC2 had the highest expression level in roots,followed by leaves,twigs,immature pods and flower buds in the plants growing in the wild.3.The plant expression vector p1302-35S-AmNAC4 was constructed and used for the transformation of the leaf discs from poplar aseptic seedlings by the Agrobacterium-mediated method.Forty one regenerated hygromycin-resistant seedlings were obtained.Through PCR and RT-q PCR analyses,seven transgenic lines of AmNAC4 were confirmed and three of them showed high expression level of the transgene.Further analyses showed that the transgenic seedlings had increased plant height,fresh weight and chlorophyll content but decreased electric conductivity and anthocyanin content compared with their control wild-type seedlinghs under salt stress.The results indicate that constitutive expression of AmNAC4 improved salt tolerance of the transgenic poplar lines.4.By transformation of rice calli with the A.tumefaciens strain containing plant expression vector p3300-35S-AmNAC4,twenty one regenerated phosphinothricin-resistant plants（the R₀generation）were obtained and their seeds（the T₁generation）were harvested.By PCR and RT-q PCR detections,twelve independent AmNAC4 transgenic lines were confirmed and six of them had high expression level of the transgene.This paper provides evidence and experimental materials for exploring the function and utilization value of the AmNAC2 and AmNAC4 genes in plant stress tolerance.