| Soil salinization had become one of the important factors limiting the growth and development of forest trees.The salt tolerance of forest trees could be improved by introducing foreign genes into cells and integrating them into the forest genome by genetic engineering technology.Plant salt-tolerant transcription factors could significantly increase plant salt tolerance by regulating the expression of downstream stress-tolerant genes.By studying the stress response and regulation mechanism of transcription factors,it could provide theory and experiment for salt-tolerant breeding of forest trees.In this study,the transcription factor Pn HB7 gene from Populus nigra L.was cloned and analyzed by bioinformatics to obtain the main structural information of Pn HB7 gene and HD-Zip family.Genetic engineering technology was used to construct genetic transformation vectors;the tobacco transgenic plants were obtained by Agrobacterium-mediated genetic transformation,and the transgenic plants were tested for salt tolerance,promoter stress response,subcellular localization and transcriptome sequencing,and the expression position,salt tolerance,stress response and forecast information of regulation pathway of Pn HB7 gene were obtained.The main results were as follows:（1）The poplar HD-Zip family consisted of 63 transcription factors,divided into four subgroups I,II,III and IV,containing 22,17,8 and 16 transcription factors,respectively.The Pn HB7 transcription factor belonged to the poplar HD-Zip family I subfamily and contained a homeodomain（HD）and a leucine zipper domain（LZ）;The gene was located on chromosome 14 of Poplar,with a length of 717 bp,encoding a polypeptide chain of 238amino acids with a molecular formula of C1185H1861N329O387S8,a molecular weight of 27165.26 Da,no signal peptide in the protein;the upstream promoter region contained a plurality of cis-acting elements associated with the stress response.（2）The PnHB7 gene was constructed on p BI121 and p CAMBIA1302 plasmids respectively,the 1 020 bp sequence of the upstream promoter region of Pn HB7 gene was constructed on p BI121 plasmid.After PCR and restriction enzyme digestion,Pn HB7 gene overexpression vector,Pn HB7:GFP fusion vector expression and promoter vector of Pn HB7 gene were obtained;the wild type tobacco was genetically transformed by Agrobacterium-mediated transformation,and the transgenic lines were obtained after screening by kanamycin;the transgenic tobacco seedlings were transplanted into the greenhouse,DNA and cDNA were extracted for PCR detection,and a bright and single target band was obtained.It was confirmed that 4 transgenic tobacco lines and 3 promoter-transformed tobacco lines were obtained.（3）The GFP fusion expression vector was transiently transformed into wild-type tobacco to carry out subcellular localization of Pn HB7 gene with empty GFP as control.The localization results showed that Pn HB7:GFP fusion protein was localized in the nucleus.（4）The results of salt tolerance test of transgenic tobacco potted plants showed that the growth and physiological indexes of overexpressed tobacco under 0.4%and 0.8%Na Cl were better than those of wild type tobacco.Under Na Cl treatment,the average diameter,biomass,abscisic acid content and proline content of transgenic plants were higher than wild type tobacco;the average MDA content,relative conductivity,the decrease rates of chlorophy II a and total chlorophy II content were lower than that of wild type tobacco.Fluorescence parameters were also better than wild-type tobacco,with higher photosynthetic efficiency and photoprotection mechanism,indicating that over-expressed tobacco had stronger salt tolerance.（5）Stress response test of Pn HB7 promoter transformed tobacco seedlings showed that tobacco seedlings could be stained with blue by GUS dye solution after treating with 2mg·L-1 aqueous solution of abscisic acid,150 mg·L-1 PBST solution of ethephon,15 mg·L-1 aqueous solution of methyl jasmonate and 0.4%aqueous solution of Na Cl for 30 minutes,indicating that Pn HB7 gene could respond to abscisic acid,ethephon,methyl jasmonate and sodium chloride（Na Cl）.（6）Transcriptome sequencing of transgenic tobacco showed hormone-related protein genes（abscisic acid,gibberellin,ethylene,salicylic acid）,oxidase genes（oxidase,peroxidase,polyphenol oxidase）,transcription factor protein gene（MYB class,WRKY,AP2,HD-Zip）and etc stress-related genes were significantly up-regulated,and some of them were located in the signaling pathway of ABA transduction.It was speculated that Pn HB7 gene plays a role in the ABA regulatory pathway. |
