Function Analysis Of Ammonium Transporter CsAMT1.3 Gene In Tea Plants

Nitrogen is one of the essential nutrients for plant growth and development,and it is also the most key factors limiting plant growth,crop yield and quality.Ammonium nitrogen and nitrate nitrogen are the two main forms of inorganic nitrogen in soil.Tea plants are very important economic crops in China,and one of the main sources of income for people in mountainous areas.As an ammonium-loving plants,tea plants have low nitrogen utilization efficiency,and nitrogen metabolism of tea plants directly affects tea quality.Therefore,how to improve the utilization rate of nitrogen fertilizers,especially ammonium nitrogen fertilizers,and improve tea quality become one of the key issues to achieve high-quality and high-yield for tea gardens.The ammonium transporters（AMT）is mainly responsible for absorption and transport of NH₄⁺in plants,so AMT play an important role in the absorption,transport and metabolism of nitrogen in tea plants.In this study,the members of the ammonium transporter family in tea plants were identified through the tea plants genome analysis,and one gene CsAMT1.3in AMT family was cloned.On this basis,the gene function was further studied.The main results are as follows:1.A total of 16 AMT family genes were identified in the genome of Camellia sinensis var.sinensis by bioinformatics analysis.All CsAMTs genes were divided into three groups according to their domains and phylogenetic tree,and all the genes were distributed on 12 chromosomes.The promoter cis-elements that might regulate the CsAMTs were predicted,and it was found that the expression of AMT in tea plant was regulated by various factors,such as environmental stress and phytohormones.According to public transcriptome data,it was found that most CsAMTs were mainly expressed in young leaves and roots.Drought,salt and Me JA stress all affected the expression of CsAMTs.2.The tea plants CsAMT1.3 was cloned.The full length of the gene is 1389 bp,without introns,encoding 462 amino acids.The protein molecular weight is 49.73 k Da and the isoelectric point（p I）is 4.95.CsAMT1.3 belongs to the AMT1 subfamily according to the phylogenetic analysis.The protein does not contain signal peptide and is a hydrophobic protein with 9 transmembrane structures and multiple phosphorylation sites.The gene was located on the cytoplasmic membrane and was mainly expressed in the shoots,with the highest expression level in the first and second leaves,and relatively low expression level in roots and vascular tissues.3.The expression level of CsAMT1.3 was different among different tea varieties,which may be related to its ammonium transport and assimilation ability.The CsAMT1.3 was not only regulated by ammonium,but also affected by photoperiod.The expression of CsAMT1.3 in the daytime was higher than that in the night,showing a typical circadian rhythm.CsAMT1.3 also showed a similar trend with the expression of glutamine synthase（GS）and glutamate dehydrogenase（GDH）in tea plant,suggesting that CsAMT1.3 may be related to nitrogen assimilation and carbon-nitrogen balance.4.When studying the effects of different nitrogen sources on CsAMT1.3 and carbon and nitrogen metabolism,it was found that the contents of tea polyphenols and catechins representing carbon pools increased when only NO₃^-and no nitrogen supply,and the expression of most genes related to catechins synthesis also showed an up-regulation trend.Compared with NO₃^-as the sole nitrogen source,both NH₄⁺and mixed nitrogen treatments had a positive effect on the accumulation of amino acids,especially theanine,glutamate and arginine.The expression of ammonium assimilation genes in nitrogen metabolism was also increased with the supply of NH₄⁺.Under the mixed nitrogen treatment,the ratio of total polyphenols to free amino acids was between sole NH₄⁺and NO₃^-supply,indicating that the carbon and nitrogen metabolism of tea plants tended to be more balanced under mixed nitrogen treatment.Under the four treatments of no nitrogen supply,sole NO₃^-or NH₄⁺supply and mixed nitrogen supply,the expression of CsAMT1.3 in different tissues was the highest when only NH₄⁺supply,and the expression of CsAMT1.3 in mature leaves was significantly higher than the other three treatments,indicating that CsAMT1.3 may be more inclined to play the role of assimilation and maintaining carbon and nitrogen balance in mature leaves of tea plants.5.Yeast functional complementation experiments prove that CsAMT1.3 can transport not only NH₄⁺but also and methylammonium,a homology of ammonium.At the same time,CsAMT1.3 could make recombinant yeast grow at low p H value,suggesting that it may be regulated by external protons.The physiological phenotypes,biochemical indicators and gene expression of CsAMT1.3 overexpressed Arabidopsis thaliana were analyzed.The results showed that transgenic Arabidopsis plants had better growth potential,more developed lateral roots,and increased contents of chlorophyll and free amino acids.Meanwhile,the expression of phosphoenolpyruvate carboxylase（PEPC）and glutamate synthase（GLT）,key genes of carbon and nitrogen metabolism,were up-regulated in transgenic plants under 4 m M NH₄⁺treatment.The results suggest that CsAMT1.3 may be induced by ammonium,which can promote nitrogen metabolism and accelerate ammonium assimilation.