Study On Nitrogen Responsed Genes PtSUT3 And PtSUT4 Involved In Wood Cellulose Synthesis In Populus Trichocarpa

Sucrose is the main photosynthetic assimilation product.Sucrose transporters（sucrose transporters or sucrose/H⁺cotransporters,SUTs or SUCs）are involved in the transport of sucrose in the apoplast,and play a key role in the transport and distribution of sucrose in plants.In this study,5 PtSUTs genes were identified and bioinformatic analysis was performed in Populus trichocarpa.PtSUT3 and PtSUT4 genes were screened by RT-qPCR,which were highly expressed in stems and regulated by nitrogen.The subcellular localization of PtSUT3and PtSUT4 were identified.The overexpression vectors and CRISPR/Cas9 gene knockout expression vectors of PtSUT3 and PtSUT4 were constructed.Genetic transformation of P.trichocarpa by Agrobacterium tumefaciens-mediated.The overexpression plants of PtSUT3and PtSUT4 genes were obtained,and the physiological and biochemical indexes were determined.The main findings are shown below:（1）The 5 PtSUTs genes were identified in P.trichocarpa,and encode polypeptide chains composed of 510-600 amino acid residues.The 5 PtSUTs proteins could be divided into three evolutionary branches,all of which contain MFS domains.Except for PtSUT4 and PtSUT6,which contain 10 and 11 transmembrane domains,the other proteins all contain 12transmembrane domains.PtSUTs proteins contain 8 identical conserved motifs.PtSUTs genes are distributed on 5 chromosomes.Segment duplication is the main factor for the expansion of PtSUTs family.（2）The results of RT-qPCR detection showed that the expression of PtSUTs gene was tissue-specific,and the expression level in the stem was higher and the expression level was regulated by nitrogen concentration.PtSUT3 and PtSUT4 genes were screened by RT-qPCR,which were highly expressed in the stem and regulated by nitrogen.The subcellular localization vector of PtSUT3 and PtSUT4 were constructed.The identification results showed that the PtSUT3 and PtSUT4 proteins were localized in the cell membrane and tonoplast membrane,respectively.（3）The overexpression vectors of PtSUT3 and PtSUT4 were constructed that were driven by the developmental xylem-specific promoter DX15.Four PtSUT3 overexpressing transgenic lines and five PtSUT4 overexpressing transgenic lines were obtained by genetic transformation of P.trichocarpa.After RT-qPCR identification,the lines S3-1,S3-2,S3-3 with a large amount of PtSUT3 gene expressed in the stem and the lines S4-1,S4-2,and S4-3 with a large amount of PtSUT4 gene expressed in the stem were screened for subsequent tests.The CRISPR/Cas9knockout expression vectors of PtSUT3 and PtSUT4 genes were constructed,and three PtSUT3gene knockout-resistant plants and five PtSUT4 gene knockout-resistant plants were obtained.（4）The wild-type plants,PtSUT3 overexpression transgenic plants and PtSUT4overexpression transgenic plants were treated with different concentrations of nitrogen（0.1,1,5 mmol/L NH₄NO₃）.The result of physiological and biochemical index determination showed that,compared with the wild type,the plant height,ground diameter and chlorophyll content of PtSUT3 and PtSUT4 genes overexpressed transgenic plants were significantly increased under three concentrations of nitrogen treatment.And the contents of soluble sugar,sucrose,glucose,fructose,starch,soluble protein and free amino acid were significantly increased in the overexpressing transgenic plants.The expression levels of sucrose metabolism-related genes（SUS1,SUS2,SUS3,VIN2,VIN3,CIN3,CIN4,NIN8/12,NIN9/11,SPS5,SPS6）were significantly up-regulated.Under normal nitrogen treatment conditions,the nitrate nitrogen content of overexpressed plants was significantly increased.Under high nitrogen treatment conditions,the nitrate nitrogen content of PtSUT3 overexpression transgenic strains S3-2,S3-3and PtSUT4 overexpression transgenic strains S4-3 were significantly reduced compared with wild type.Under the three nitrogen treatment conditions,there was no significant difference in ammonium nitrogen content between overexpressed plants and wild type.（5）The results of histochemical staining and scanning electron microscopy showed that,the wood xylem region was broadened and the secondary cell wall was thickened in the PtSUT3 overexpressing transgenic plants and PtSUT4 overexpressing transgenic plants compared with the wild type.Compared with wild type,hemicellulose and cellulose contents were significantly increased and lignin content was significantly decreased in overexpressed plants under three nitrogen treatment conditions.In addition,the cellulose synthesis gene（Ces A4,Ces A7,Ces A8）were significantly up-regulated,and the expression levels of lignin synthesis-related genes（PAL2,C4H1,C3H2,4CL1,COMT2 and CCo AOMT1）were significantly down-regulated in overexpressed plants.This suggests that overexpression of PtSUT3 and PtSUT4 genes can promote the synthesis of wood cellulose.These results lay an experimental basis for further identification of the functions of sucrose transporter genes PtSUT3 and PtSUT4 in the synthesis of wood secondary cell wall cellulose,and also provide a theoretical basis for the cultivation of poplar germplasm through genetic improvement and rational application of nitrogen fertilizer.