Screening Of Different Nitrogen Form Response Genes Of Populus×xiaohei T.S.Hwang Et Liang And Analysis Of Transcriptional Characteristics Of Over-expressing AlaAT Plants

Nitrogen is one of the important elements that affect the growth and development of plants.Plants mainly absorb inorganic nitrogen in the soil through their roots,including ammonium nitrogen and nitrate nitrogen.Different forms and concentrations of nitrogen have different effects on physiological and biochemical levels of plants,but its molecular regulation mechanism is currently unclear.In this study,used hydroponic wild-type Populus × xiaohei T.S.Hwang et Liang as experimental materials,and it was treated with different forms and concentrations of nitrogen.RNA-Seq sequencing technology was used to screen the genes that respond to different nitrogen forms in the roots of Populus×xiaohei T.S.Hwang et Liang;the relative change coefficient method was used to determine the response degree of root nitrogen metabolismrelated genes,and it was found that the relative change coefficient of AlaAT gene(AlaAT3)was the largest.In order to further study the role of alanine aminotransferase(AlaAT)in different nitrogen forms treatment,we found over-expressing AlaAT gene transgenic line from the transgenic plant bank preserved in our laboratory,and adopted the transformant line 13 as the test material.Bioinformatics methods such as GO enrichment and KEGG enrichment were used to analyze the differential genes and study their influence on nitrogen response.The results are as follows:(1)Characteristic modules produced by the wild-type roots of Populus×xiaohei T.S.Hwang et Liang under different forms and concentrations of nitrogen,the regulatory network analysis was performed,and the transcription factors and functional genes of ammonium nitrogen and nitrate nitrogen specific response are found.The transcription factor of ammonium nitrogen-specific response is KAN1,WRKY70,ERF9,ect,the functional gene of the specific response is HRGPs,RLKs,etc,and the Nitrate nitrogen is found to MYB56 is specific response transcription factors and CIPK,LBD gene is specific response functional gene.(2)A co-expression network of nitrogen assimilation genes under different forms of nitrogen treatment was constructed,and the relative change coefficient analysis was carried out.The ranking result was AlaAT>AS>GDH>GS>GOGAT.It is speculated that AlaAT gene may play an important role in nitrogen response.(3)Different form nitrogen treatment wild type and AlaAT overexpressing transgenic line,the detection of physiological index,the Nitrate reductase(NR),Nitrite reductase(NIR),chlorophyll,and free amino acid is significantly higher than the wild type.(4)RNA-Seq sequencing results showed that under the treatment of 3.0 mM ammonium,2638 and 7835 differentially expressed genes were obtained in the leaf and root tissues of the transformant 13 and the wild type;Under the treatment of 3.0 mM nitrate,7316 and 6421 differentially expressed genes were obtained in leaf and root tissues.(5)GO enrichment and KEGG enrichment analysis of the above differentially expressed genes showed that,these genes are mainly involved in biological processes such as metabolic process,binding,catalytic activity,and cellular processes,and there are 49 significantly enriched metabolic pathways,mainly related to metabolic pathways and phenylpropane biological pathways.synthesis,starch and sucrose metabolism,plant-pathogen interaction,etc.In this study used cross-tabulation and relative change coefficient methods to screen out transcription factors and functional genes that may respond specifically to the nitrogen form response of Populus×xiaohei T.S.Hwang et Liang,and the degree of nitrogen metabolism related genes affected by nitrogen form was sequenced,and it was found that AlaAT gene was most affected by nitrogen form.RNA-Seq analysis showed that the gene expression patterns in the roots and leaves of overexpressing AlaAT changed significantly under the treatment of differential nitrogen forms.These results laid an important foundation for revealing the function of AlaAT gene in poplar plants response to different forms of nitrogen,and provided a new idea for exploring the molecular mechanism of nitrogen forms affecting plant changes.