The Molecular Mechanism Of BpNAC090 Transcription Factor Conferring Drought Tolerance In Betula Platyphylla

The NAC transcription factor（TF）family is a specific transcription factor in plant,which contains a large number of members.NAC transcription factors mainly participate in many biological processes in regulating growth and development of plants,such as external environment stress response,plant morphosis,plant senescence regulation,fruit development and so on.This research studied the tolerant function of BpNAC090 gene under drought stress in Betula platyphylla Suk.,which was found in the first layer of birch hierarchical gene regulatory network.The main results are as followings:（1）20%PEG6000 was used for simulation of drought condition.The analysis of BpNAC090gene expression pattern showed that BpNAC090 was upregulated expressed in birch,and expression level of BpNAC090 was highest at 3 h and then decreased.However,the expression of BpNAC090 was upregulated under drought stress.At the same time,BpNAC090 was mainly expressed in roots and leaves of birch.These results indicate that BpNAC090 gene plays important roles in response to drought stress in birch.（2）Overexpressed and gene-knockdown BpNAC090 vectors were constructed and transformed into birch using Agrobacterium tumefaciens-mediated transformation.Reverse transcription–quantitative polymerase chain reaction（RT-q PCR）was used to analysis of expression level of overexpressing lines（OE）.OE1,OE2 and OE5 had higher expression levels and were selected for Western Blot to identify whether they could successfully express.Gene-knockdown transgenic lines（nac1,nac2 and nac3）were obtained used for further analysis based on CRISP-Cas9 system.OE and gene-knockdown transgenic lines were treated with PEG6000and the wild-type（WT）birches were set as control.After drought stress,parts of OE-lines（OE1,OE2 and OE5）leaves got yellow but still alive;while wild-type and gene-knockdown birches dried rot and parts of leaves fell off.Then watered for 12 d,OE1,OE2 and OE5 recovered growth but wild-type and gene-knockdown birches did not.The results that BpNAC090 could reduce cell damage and enhance drought stress tolerance of OE lines by reducing MDA content,ROS content,H₂O₂ content and electrolyte leakage,improving POD and SOD activity and increasing proline content.（3）BpNAC090 could directly upregulate 51 genes and downregulate 66 genes though the combined analysis of RNA sequencing（RNA-Seq）and chromatin immunoprecipitation sequencing（Ch IP-Seq）under drought stress.BpNAC090 could bind to EOMES2（“AACACC”）,ABRE（“CACGTG”）and Tgif2（“TGTCA”）motifs by yeast one hybrid（Y1H）and electrophoretic mobility shift assay（EMSA）,which could response to drought stress.Eleven genes were seldomly selected from 51 upregulated genes of Ch IP-PCR and Ch IP-q PCR and could be bound in promoter by BpNAC090,of which 5 genes had drought stress tolerance by physiological indicators after transient transformation.（4）BpNAC090 could bind with EOMES2,ABRE and Tgif2 motifs to regulate downstream target genes under drought stress,which was regulated by BpNAC090 phosphorylation.Serine only at position 205 of BpNAC090 can be phosphorylated by drought treatment and make BpNAC090 have drought stress tolerance.The amino acid residues of Ser205 were substituted with Ala(S²⁰⁵A)and the mutated BpNAC090 gene was stably transformed into birch.The loss of phosphorylation of S²⁰⁵A could not restore drought tolerance capability in mutant birch because the ability binding to EOMES2,ABRE and Tgif2 motifs got weaker.These results suggested that phosphorylation of S205 is necessary for BpNAC090’s ability to confer drought tolerance.（5）BpNAC090 did not have self-activation activity,could interact with protein kinase Bp SRK2A and be phosphorylated by Bp SRK2A.（6）A new identification technique of DNA-binding protein（R-Ch IP–d Cas9）was developed and upstream regulators of BpNAC090 was found based on R-Ch IP–d Cas9,including Bpb ZIP16,Bp C3H14,Bp AGL11,Bp WHY1 and Bp Ge BP TFs.These 5 TFs could bind to promoter region of BpNAC090 and regulate BpNAC090 via Ch IP and RT-PCR analysis.This research reveals the molecular mechanism of drought tolerance regulated by BpNAC090 in birch and provides vital information for molecular mechanism of drought tolerance in birch and creating highly-resistance birches.