| Carbohydrates play a very important role in the life activities of plants.They not only provide energy for the growth and development of plants as energy substances,but also supply intermediate products for the synthesis of proteins,nucleic acids and lipids through some metabolic pathways.In addition,sugar is also used as a signal molecule to participate in the physiological regulation of plants.Plant hexokinase(HXK)is a type of multifunctional protein that can not only phosphorylate hexose,act as a sugar sensor as well.When the internal sugar level rises,it can inhibit the expression of some photosynthetic-related genes and regulate cellular metabolism and some sugar-related pathways.Increasing evidence shows that plant hexokinase plays an important role in regulating growth and development,while the function of poplar hexokinase is poorly understood.In this study,poplar was used as the research material to analyze the hexokinase gene family and study the gene function of HXK1.The analysis of the hexokinase gene family(six hexokinase genes,Ptr HXKs)of Populus trichocarpa includes some basic characteristics: sequence characteristics,expression patterns,protein localization and kinase activity.Secondly,through poplar transgenic technology,gene function remediation detection technology,transcriptome analysis technology,and yeast two-hybrid technology,the function of 84 K poplar PagHXK1 gene in regulating plant growth was studied.The main results are as follows:(1)The analysis of the protein sequence characteristics of the Populus trichocarpa hexokinase gene family showed that the poplar hexokinase is divided into two types: Type A(Ptr HXK3)type and Type B(Ptr HXK1-2,Ptr HKL1-3)type.The analysis of promoter cis-elements showed that poplar hexokinase may be involved in some response processes such as stress,hormones and light.(2)The six genes of Populus hexokinase gene family were cloned in vitro,and the poplar HXK-GFP fusion protein expression vector was constructed and the tobacco transient transformation experiment was performed.Fluorescence observation results showed that Ptr HXK1-2 and Ptr HKL1-3were localized in mitochondria while Ptr HXK3 were localized in the chloroplast.(3)The yeast expression vector of poplar hexokinase full-length and truncated hexokinase with the transmembrane domain or signal peptide removed was constructed to transform the yeast mutant YSH7.4-3C.The results of yeast phenotype analysis after transformation showed that Ptr HXK1-3protein has stronger kinase activity than Ptr HKL1-3 protein,and the transmembrane domain or signal peptide domain has a greater impact influence on the kinase activity of Ptr HXK1-3,but it has less effect on Ptr HKL1-3 protein.Moreover,using glucose and fructose as substrates,the affinity of Populus hexokinase in Populus to different substrates was tested in vitro.The results showed that compared with fructose,hexokinase protein in Populus has a higher affinity for glucose,and the affinity of Ptr HXK1-3to glucose is better than that of Ptr HKL1-3 protein.When fructose is used as the substrate,the Ptr HXK3 protein has the lowest affinity for fructose.(4)The analysis of tissue expression patterns of hexokinase gene family in Populus showed that the expression of Ptr HXK1,Ptr HKL1,Ptr HKL2 and Ptr HKL3 is higher in leaves,Ptr HXK3 is higher in roots,and Ptr HXK2 is higher in immature xylem.Among them,the expression patterns of Ptr HKL2 and Ptr HKL3 genes are very similar.In addition,the results of the detection of hexokinase expression levels at different stages of poplar callus differentiation showed that during the process of callus differentiation,the expression level and enzyme activity of the poplar hexokinase gene family increased to a certain extent,and the expression level of Ptr HKL2 changed most significantly,indicating that poplar hexokinase may be involved in the regulation of callus differentiation.(5)In 84 K poplar(Populus alba × Populus glandulosa),PagHXK1 gene homologous to Arabidopsis At HXK1 gene was cloned to construct an overexpression vector to transform 84 K poplar.Phenotypic analyses showed that overexpression of PagHXK1 gene can inhibit poplar growth,and reduce the net photosynthetic rate and stomatal conductance,negatively regulate the expression of photosynthesis-related genes.Transcriptomics analysis of transgenic plants and wild-type plants showed that the functional enrichment analysis of differential genes GO and KEGG respectively showed that 36 and 51 differential genes were enriched in the photosynthesis pathway.(6)The overexpression vectors 35S:PagHXK1 and 35S:PagHXK1-G104 D encoding the loss of hexokinase activity were constructed and transformed into the Arabidopsis gin2-1 mutant.The results showed that under 6% glucose treatment,overexpression of PagHXK1 and PagHXK1-G104 D can remedy the glucose-insensitive phenotype of the gin2-1 mutant,and under high light conditions(increased sugar accumulation),the transgenic lines can replenish their weak growth phenotype,indicating that the PagHXK1 gene has a similar function to the Arabidopsis GIN2/HXK1 gene.(7)The membrane system yeast two-hybrid library was constructed with 84 K poplar as the material,and the interaction protein was screened with PagHXK1 protein as the bait protein.A total of48 candidate proteins were screened,of which 19 were related to the regulation of photosynthesis.Furthermore,the interaction between PagHXK1 protein and Pag OEE2(oxygen-evolving enhancer protein 2)protein was verified,and the results showed that there was an interaction between the two proteins in the yeast system,suggesting that PagHXK1 is related to the regulation of plant photosynthesis.In summary,this study conducted a detailed analysis of the Populus hexokinase gene family,and systematically studied the function of the PagHXK1 gene in regulating the growth of poplars.The present investigation lays the foundation for the in-depth analysis of poplar HXK gene function,which may promote the further study of plant HXK gene function. |
PDF Full Text Request