The Function Analysis Of Ammonium Transporter Gene PsAMT1.2 In Poplar Simonii

Nitrogen is one of the most important elements in plant growth and development,and it is the main component of plant nucleic acid,protein,chlorophyll,etc.The main transporters（Ammonium Transporters,AMT）located on the plasma membrane.In addition,the expression level of genes encoding ammonium transporter may increase,enhance the plant’s ability to absorb and metabolize NH₄⁺under adverse condition,promote the accumulation of chlorophyll,photosynthesis and protein synthesis,etc.,thereby maintaining the plant normal growth and development and metabolic processes.The experiment selected the salicylate-tolerant and infertile tolerant Poplar simomii as the study maters.The function of the PsAMT1.2 gene encoding the ammonium transporter of P.simonii was verified,and the physiological indexes and nitrogen accumulation and metabolism of the transgenic poplar that overexpressed the PsAMT1.2 gene under salt stress were studied.The response of PsAMT1.2 gene to salt stress and the effect on nitrogen absorption and metabolism under salt stress are elucidated,which is helpful to elucidate the regulation mechanism of nitrogen absorption and metabolism under salt stress at the molecular level.The results are as follows:1. The PsAMT1.2 gene was expressed in the root of P.simonii.The PsAMT1.2gene was cloned from the root of P.simonii,with the length of 1761 bp and encoding586 amino acids.The sequence similarity of the cloned gene between P.simonii and Populus trichocarpa was 98%,and the gene was identified as PsAMT1.2 gene in P.simonii.The PsAMT1.2 gene promoter sequence was isolated and cloned,and the PsAMT1.2 gene promoter fragment length of 1000 bp was obtained,and the homology with the P.trichocarpa gene promoter was 75%.2. The PsAMT1.2 gene of P.simonii was transiently expressed in tobacco,and it was found that the PsAMT1.2 gene was located on the membrane,indicating that the PsAMT1.2 protein is active on the membrane and is a membrane transport protein.The promoter sequence was connected to an expression vector with a deleted promoter and a GUS tag,and the PsAMT1.2 gene promoter was introduced into Arabidopsis thaliana by a dipping method.GUS staining homozygous transgenic A.thaliana showed that PsAMT1.2 has transcriptional activity in the root system.3. Heterologous expression of the PsAMT1.2 gene restored the growth activity of high-affinity ammonium transporter-deficient yeast 31019b at low concentrations of expressing the PsAMT1.2 gene conformed to the double regression curve fitted by the saturated Michaelis-Menten equation,Km was 80.603±8.019.4. Three PsAMT1.2-overexpressing transgenic lines were obtained.After 21 days of salt treatment,the physiological indexes of transgenic poplars were measured,and it was found that the PsAMT1.2-overexpressing transgenic poplars plants under salt stress had increased chlorophyll content,net photosynthetic rate and plant height difference compared with wild type.The relative ¹⁵N content of transgenic poplars wild type,indicating that overexpression of PsAMT1.2 is beneficial to the uptake of NH₄⁺by transgenic poplars under salt stress.5. The experiment found that the PsAMT1.2-overexpressing transgenic poplars and roots,decreased NO₃^-content in roots,and glutamate synthase in leaves and glutamine synthesis in root enzyme activity is enhanced,leading to the accumulation of glutamate in the leaves and glutamine in the roots compared to the wild type.In addition,the expression level of glutamine synthetase gene GS1.3 and glutamate synthetase gene Fd-GOGAT was increased in the root and leaves of PsAMT1.2-overexpressing transgenic poplar under salt stress,the glutamine content in the root and glutamate content in the leaves was increased compared to the wild type,accumulating sufficient substrate content required for protein synthesis for poplar growth.