Preliminary Research On The Functions Of PagGS1.3 And PagNADH-GOGAT Genes In Populus Alba×p.glandulosa

Glutamine synthase（GS）and glutamate synthase（GOGAT）are the key enzymes for nitrogen assimilation in plants.Nitrogen is a basic nutrient element necessary for plant growth and development,and more than 95%of NH₄⁺in higher plants is assimilated through the GS/GOGAT cycle.In this study,PagGS1.3 highly expressed in stems and regulated by nitrogen was identified.84K poplar（Populus alba×P.glandulosa）was used as the research material,and the developing xylem-specific overexpression transgenic material（OE-PagGS1.3）and knockout mutants（Paggs1.3）of the PagGS1.3 gene were created respectively.Differences in phenotype,physiology,biochemistry and wood properties were analyzed between PagGS1.3 transgenic materials and wild-type（WT）under low nitrogen,normal nitrogen and high nitrogen treatments.At the same time,double mutant materials of PagNADH-GOGAT（Pagnadh-gogat）were created,and the differences in phenotype,physiology and biochemistry of the double mutant and WT plants were preliminarily compared.The main findings are as follows:（1）PagGS1.1 and PagGS2 were highly expressed in leaves;PagGS1.2 was highly expressed in roots;PagGS1.3 was highly expressed in stems.The expression level of PagNADH-GOGATs was highest in roots,followed by stems;PagFd-GOGATs were highly expressed in leaves.Except for PagGS1.2,the other members in GS and GOGAT gene family were all regulated by N in their highly expressed tissues.（2）The p ROKⅡ-DX15::PagGS1.3 recombinant expression vector and the CRISPR/Cas9knockout vector of PagGS1.3 were constructed,and genetically transformed into 84K poplar.PagGS1.3-overexpressing lines and Paggs1.3 mutant lines were obtained.（3）After treatments with different nitrogen concentration,the transcription level of GS1.3was enhanced;the activities of GS and GOGAT enzymes increased;the contents of free amino acids and soluble proteins increased;the content of NH₄⁺decreased,whereas the mutants exhibited an opposite trend.Under high nitrogen,it also increased the content of soluble sugar and starch.Lower lignin content and higher cellulose content were determined in OE-PagGS1.3.At the same time,the expression levels of lignin monomer synthase genes were down-regulated to different degrees;the expression levels of cellulose synthase genes were also increased to different degrees;the mutants showed the opposite trend.（4）Pagnadh-gogat double mutants were created based on CRISPR/Cas9 technology.Compared with WT,mutants showed obviously slow growth and severely damaged root growth;GS and GOGAT enzyme activities,the contents of free amino acids and soluble protein significantly declined,while the contents of NH₄⁺,NO₃^-,soluble sugar and starch significantly accumulatated.In conclusion,PagGS1.3 and PagNADH-GOGAT genes play important roles in nitrogen metabolism,and PagGS1.3 could cause the expression changes of genes related to lignin and cellulose synthesis,afterwards affecting biosynthesis of poplar lignin and cellulose.These results lay an experimental basis for further characterizing the functions of PagGS1.3 and PagNADH-GOGAT proteins,clarifying the roles of GS/GOGAT cycle in nitrogen metabolism and wood formation.This study also provided a theoretical basis for obtaining forest varieties with eminent material quality through genetic improvement and rational application of nitrogen fertilizers.