Response Of Cytokinin Synthesis Gene IPT5 To Low Temperature Stress In Poplar

Low temperature restricts the growth and development of plants and their regional distribution.Using genetic engineering technology can improve the cold resistance of poplar,expand its suitable survival area to a certain extent,and expand the scope of introduced cultivation.In this study,bioinformatics analysis of the isopentenyltransferase gene of Populus trichocarpa.Since Populus trichocarpa could not be planted in China,based on the existing transformation platform of Populus hopeiensis in our laboratory,we used plant overexpressed vector carrying Pt IPT5 gene for genetic transformation of Populus hopeiensis by Agrobacterium-mediated leaf disk method and obtained transgenic lines.Identification and expression of Pt IPT5 gene in transgenic lines at DNA,transcriptional and metabolic levels using PCR,fluorescence quantitative PCR,low temperature stress test and cytokinin-like metabolite assay,respectively.The main results are as follows:1.Cloning and bioinformatics analysis of Pt IPT5 gene in Populus trichocarpa.The Pt IPT5 gene was cloned,and bioinformatics analysis revealed that the full-length CDS sequence of the coding region of this gene is 984 bp and can encode 327 amino acid residues.Its promoter region contains multiple responsive cis-acting elements.Pt IPT5 protein is a hydrophilic stable protein with no signal peptide and no transmembrane structure,and has Cas3＿I superfamily structural domain.Closer kinship and evolutionary relationship of IPT proteins with Populus deltoides and Salix sinopurpurea.Protein subcellular localization predicted in chloroplasts.2.Genetic transformation of Populus hopeiensis using Agrobacterium-mediated leaf disk method was screened for thaumatin,and a total of seven resistant lines were obtained,and their total DNA was extracted and tested by PCR.The results showed that all seven transgenic lines contained the target gene bands,while the non-transgenic lines did not amplify any bands,tentatively proving that the target gene had been inserted into the genome of Populus hopeiensis.3.The seven transgenic lines were further tested by fluorescence quantitative PCR,and the results showed that the fluorescent signal of Pt IPT5 gene was detected in all transgenic lines,and the expression of transgenic lines was higher than that of non-transgenic lines.The expression of Pt IPT5 gene in the transgenic lines was about4.31-8.30 times higher than that in the non-transgenic lines,with the highest expression in lines 1 and 2,which were 8.30 and 7.55 times higher than that in the non-transgenic lines,respectively.4.Seedlings of transgenic lines 1 and 2 with high expression by fluorescence quantitative PCR were selected for preliminary cold tolerance tests.Determination of low temperature related physiological indicators such as relative conductivity,chlorophyll,photosynthetic rate,soluble protein,peroxidase,superoxide dismutase and malondialdehyde in leaves of each lines.The results showed that the two transgenic lines showed a more pronounced tolerance to low temperature stress compared to the non-transgenic lines.5.Qualitative and quantitative determination of cytokinin-like metabolites using ultra performance liquid chromatography and tandem mass spectrometry.Significant increase in the content of cytokinin-like metabolites in leaves of Populus hopeiensis transgenic for Pt IPT5.Low temperature decreased the content of this metabolite,but the metabolite content in the transgenic lines was still significantly higher than in the non-transgenic control.The Pt IPT5 gene mainly regulates the isopentyl dilute pyrophosphate cytokinin metabolic pathway and synthesizes t Z,i P and DZ-type cytokinins.In this study,the sequence of Pt IPT5 gene was cloned and obtained.Through the systematic analysis of genes and low temperature stress tests on transgenic lines,the molecular characteristics of the gene and its encoded protein were initially understood,and the biological functions involved in Pt IPT5 gene and its response to low temperature stress were explored,which laid the foundation for further clarification of the function of IPT gene and is of great significance to improve the low temperature tolerance research of Populus hopeiensis.