| Poplar is one of the main tree species of artificial forest in China,which has important economic value and ecological benefit.However,compared with other tree species,poplar is a specie with high water consumption.Large-scale planting of poplar plantations in arid and semi-arid areas with poor water resources will not only have a low survival rate of trees,but also may exacerbate the shortage of water resources in the region.Therefore,cultivating new poplar varieties with strong drought resistance and water use efficiency is an urgent problem to be solved to ensure the construction of artificial forests and ecological security in China.The stomatal are the channels for the plant to exchange gas and lose water with the external environment.By adjusting stomatal movement,optimizing transpiration rate and photosynthesis rate is of great significance for improving the drought resistance and water use efficiency of poplars.In this study,we cloned the stomatal aperture regulation gene PagHCF06 from poplar（P.alba×P.glandulosa）,and investigated the expression patterns of this gene under different tissues and stresses.Through drive the expression of the GUS reporter gene by different PagHCF06 gene promoter deletion fragments,analyze the active region of the PagHCF06 gene promoter;By cultivating Arabidopsis lines transfected with PagHCF06 gene and poplar mutant lines that target to edit the promoter of PagHCF06gene mediated by CRISPR-Cas9,the mechanism of PagHCF06 gene in regulating stomatal aperture and drought resistance was analyzed.The main results were listed as follows:1.Cloning and expression pattern analysis of PagHCF106 gene（1）The CDS sequence of PagHCF106 is 792 bp in length and encodes 251 amino acids.（2）Phylogenetic tree analysis showed that the PagHCF106 gene was evolutionarily conserved compared with that of other species,and evolution from the same branch node,and both contain the Mtt＿hcf106 superfamily domain.This information indicates that PagHCF106 may be conservative in function.（3）Tissue specificity and stress-induced expression patterns of PagHCF106 were analyzed by q RT-PCR.The results showed that the expression level of PagHCF106 was higher in mature leaves and phloem.In addition,its expression level was strongly induced by salicylic acid（SA）,methyl viologen （MV）and dehydration,but the change in abscisic acid（ABA）stress is not significant（4）The results of subcellular localization showed that PagHCF106 was mainly localized to chloroplasts.2.Effects of PagHCF106 gene on the growth and drought resistance of Arabidopsis.（1）A total of 17 Arabidopsis lines（referred as c-ox lines）overexpressing the PagHCF106 gene in hcf4 mutants were cultivated by the floral dip method, and a total of 17 T3 homozygous strains were obtained by chi-square test, genome PCR and q RT-PCR.（2）Through drought pre-experiment,the representative strains c-ox-7,c-ox-10 and c-ox-14 were selected for follow-up study.（3）The stomatal aperture of hcf4 mutant was the smallest,while the stomatal aperture of c-ox-7,c-ox-10 and c-ox-14 was similar to that of the wild-type Col-0,and the photosynthetic rate,transpiration rate and biomass of the mutant was significantly reduced,while,the biomass of wild-type,mutant and recombined strains were not significantly different after 45 days growth.（4）The mutants,wild-type and recombined strains were treated with drought stress,and it was found that under drought stress,the degree of wilting and oxidative damage suffered by the recombined strain and the wild-type were basically similar,but were more serious than the hcf4 mutant.At the same time, the water loss rate of the separated leaves of the recombined strain is also greater than that of the mutant,showing a positive correlation with the stomata aperture.3.Cloning and activity analysis of the promoter of PagHCF106 gene.（1）The promoter sequence of PagHCF106 gene was cloned from poplar（P.alba×P.glandulosa）by genoome walking and PCR technology,with a total length of 1527 bp.（2）Through Plant CARE online software analysis,it is found that the promoter contains a large number of light-responsive elements and hormone and abiotic stress-responsive elements.（3）Through truncation analysis of the promoter,a plant vector with promoter fragments of different lengths driving the GUS reporter gene was constructed and transformed into Nicotiana benthamiana transiently.The active region was found to be mainly located within-380 bp upstream area by GUS staining andβ-glycosidase activity measurement.4.Effect of PagHCF106 gene on stomatal aperture and growth of poplar（1）The CRISPR/Cas9-mediated promoter 2-target gene editing vector was constructed by enzyme cleavage ligation and overlapping PCR.（2）Through the method of Agrobacterium-mediated transformation of stem segments,genome edited poplar lines were cultivated.Through identification,17 promoter-targeted editing poplar lines（referred as CS lines）were obtained.Scanning electron microscopy found that the stomatal aperture of genome editing lines were generally lower than those of the wild type,and then the representative CS-2、CS-3、CS-8 and CS-9 for follow-up study.（3）By measuring the photosynthetic rate and water use efficiency,it was found that the photosynthetic rate of gene editing lines decreased in varying degrees,but the water use efficiency increased significantly compared with WT.（4）By measuring the water loss rate of detached leaves and hydrogen peroxide content of the guard cell,it was found that the water loss rate of CS strain was significantly lower than that of the wild type,and the hydrogen peroxide content of the guard cell in CS strain was significantly higher than that of the wild type.In summary,PagHCF106 gene and Arabidopsis HCF106 gene are functionally conserved,and it is preliminarily speculated that PagHCF106 regulation of stomatal movement is related to the accumulation of guard cell chloroplast ROS;through genome editing technology,the expression of this gene in poplar can be effectively reduced and cultivated Poplar plant with improved water use efficiency.This research lays the foundation for the cultivation of drought-resistant and water-saving poplar varieties. |
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