Functional Analysis Of Transcription Factor ChNAC1 Gene Involved In Drought Resistant In Cerasus Humilis

As an unique species in China,Cerasus humilis belongs to the Rosaceae economic tree family of Cerasus.This typical perennial plant is native to remote northern mountains,the wilderness,and some other sparsely populated areas.It exhibits a high resistance to drought,cold,and barren areas.Although C.humilis is a drought-tolerant plant,it still lacks sufficient understanding of its intrinsic molecular mechanism of drought resistance.The NAC(NAM,ATAF1/2,CUC2)transcription factor family is a plant-specific transcription factor superfamily and is found in a wide variety of plants.NAC mainly involves in plant growth,development and biotic or abiotic stress response.The research on transcription factor has been matured in Arabidopsis,rice and other model plants.However,the research of forest only focuses on regulating wood fonnation,monoterpene and anthocyanin synthesis,research on regulating abiotic stress needs to be strengthened.In order to further enrich the gene resource of forest NAC transcription factor,we cloned and characterized the NAC transcription factor ChNAC1 with C humilis cultivar "Nongda No.4' as experimental material to explore its function in response to drought stress.The main research work and results are as follows:1?The ChNAC1 gene was cloned from C.humilis leaves by using RT-PCR method.The full length of ChNAC1 cDNA was 894 bp,which encodes a protein containing 297 amino acids.It had a typical NAM conserved domain with a molecular weight of about 34.21 kDa and an isoelectric point of 8.09,which was an unstable,hydrophilic protein.Amino acid sequence alignment revealed that ChNAC1 contains a conserved NAC domain at its N-terminal and is further divided into five subdomains(A-E).The C-terminal region was highly variable.The phylogenetic tree indicated that C.humilis ChNAC1 has high homology with tobacco NtNAC and petunia PhNAC.2?Subcellular localization indicated that ChNAC1 localizes to the nucleus and yeast one-hybrid showed that the C-terminus of ChNAC1 has transcriptional activation activity,Yeast two-hybrid result showed that ChNAC1 is capable of forming homodimers in yeast cell.3?Drought resistance test on the obtained ChNAC1 overexpressing lines Line1?Line4 and WT?mutant(atafl)showed that overexpression of ChNAC1 enhances the drought resistance of Arabidopsis,which is the opposite of the homologous gene ATAF1 in Arabidopsis.Physiological indicators showed that overexpression of ChNAC1 increased relative water content,reduced ion leakage,increased chlorophyll content and leaf comprehensive performance index(PI),reduced ROS(H2O2O2-)accumulation and increased antioxidant enzymes(POD,SOD)activities,increased the contents of permeable substance(soluble protein,proline).4?ChNACl could increase the sensitivity of Arabidopsis seedlings to ABA.qRT-PCR analysis showed that ChNAC1 enchanced the expression levels of ABA signal transduction pathway genes KIN1,KIN2,RD20 and drought-related gene COR47 in Arabidopsis under drought condition,indicating that ChNAC1 relies on ABA signaling pathway.In summary,evidence from molecular,morphological and physiological experiment suggested that ChNAC1 can be used as a candidate gene to improve plant drought tolerance.