Functional Research Of PagTCP20 Genes In Populus Alba × P.glandulosa

Poplar is a model species of woody plants,with fast growth,strong adaptability,wide distribution and other characteristics,widely used in road greening and afforestation.Nitrate（NO₃^-）is an important source of nitrogen in soil,but also a key signal of root development.TCP20 is a member of TCP gene family.Previous studies had found that TCP20 played an important role in nitrate-induced system signaling,which could modulate lateral root responses to nitrate stress.Therefore,the PagTCP20 gene was cloned by wild-type 84K poplar（Populus alba×P.glandulosa）,and the overexpression vector was constructed in this study,and the84K poplar was transformed by agrobacterium-mediated leaf plate method to obtain transgenic plants.The differences and changes of PagTCP20 transgenic and wild-type strains in root development and physiological indexes were analyzed after treatment with low nitrogen（0.5m M Na NO₃）,normal nitrogen（1.0 m M Na NO₃）and high nitrogen（5.0 m M Na NO₃）,and the function of PagTCP20 gene was preliminarily explored.The results of the study were as follows:（1）PagTCP20 gene cloning and bioinformatics analysis.Using the c DNA of wild-type84K poplar tissue rooting seedlings as a template,PagTCP20 gene was cloned,and the analysis of the NCBI online website found that the PagTCP20 gene was composed of 609bp and encoded 202 amino acids.Sequence alignment analysis showed that PagTCP20 protein contained b HLH domains unique to the TCP family.（2）PagTCP20 gene overexpression study.The pROKII-PagTCP20 expression vector was constructed,and 84K poplar was infected by Agrobacterium-mediated leaflet transformation,and finally three overexpression strains were obtained,and the overexpression strains specifically increased the expression of TCP20 through the analysis of TCP20 gene expression levels.（3）Determination of root development and physiological indicators and related genes.Compared with the wild type,the root length and fresh weight of PagTCP20 transgenic plants increased significantly,the main nitrate response genes NRT1.1,NRT2.1,NIA1,NAR2.1 and Ni R were significantly upregulated,and the contents of nitrate nitrogen,ammonium nitrogen and amino acids increased.The number and average length of lateral roots increased significantly during low nitrogen treatment,and the expression of NRT1.1,NIA1 and NIA2increased significantly,while the associated genes remained almost unchanged during high nitrogen treatment.Under high nitrogen conditions,the nitrate nitrogen content in the roots of each transgenic strain increased,while the content of ammonium nitrogen and amino acid did not change much.In summary,PagTCP20 plays an important role in nitrogen metabolism of 84K poplar,which can regulate root development in response to nitrate and promote the growth of poplars.This study will provide a theoretical basis for obtaining genetically improved forest varieties and rational application of nitrogen fertilizer.