Study On The Mechanism Of Regulation Of Ammonium Nitrogen Uptake By Tea Plant CsAMT2s Genes

Tea tree（Camellia sinensis（L.）O.Kuntze）is a perennial evergreen leafy cash crop in China,and according to statistics,the area of tea plantations in China has expanded to 3,264,100 hm² in 2021,with an annual output of 3,063,200 t,ranking firmly as the first in the world.The application of nitrogen（N,nitrogen）fertilizer in tea plantations can effectively increase the nutrient supply of the soil to tea trees,promote the absorption of nutrients by the root system of tea trees,and enable tea trees to maintain normal physiological metabolism,which is of far-reaching significance to improve the yield and quality of tea leaves.As a class of ammonium-loving plants,the yield and quality of tea leaves are closely related to the uptake of ammonium nitrogen（NH₄⁺-N）.The uptake and transport of NH₄⁺-N by higher plants is mainly through the ammonium transporter protein AMT（Ammonium transporter）,and the transport of NH₄⁺across the membrane mediated by tea tree ammonium transporter protein Cs AMT is the first step for external nitrogen nutrients to enter tea tree cells,which provides a strong guarantee for the subsequent export of nitrogen assimilates.In this study,based on the previous research results of the group,we used the sequence of CsAMT2s coding sequence of DB（nitrogen efficient type）as the reference sequence,and cloned the CsAMT2s coding sequence of HD（high nitrogen efficient type）and MX（low nitrogen efficient type）to further compare and analyze the differences of their protein sequences.To investigate the correlation between root vigor,meteorological factors and CsAMT2s expression in tea plants in different seasons.The differences between nitrogen uptake and transcript levels of CsAMT2s in tea trees with different nitrogen uptake types were investigated by different fertilization treatments.The functional study of heterologously expressed Cs AMT2.3 gene was carried out in an attempt to reveal the regulatory role of Cs AMT2.3 gene in the uptake and transport of NH₄⁺in tea trees,and to provide a scientific reference at the molecular level for the rational application of nitrogen in production.The main findings are as follows.1.Cloning and analysis of CDS region of different nitrogen absorbing tea cultivars CsAMT2s genes.The coding sequences of CsAMT2s of DB was used as the reference sequence,and the sequences of CsAMT2s of HD and MX were cloned separately.Only the sequence of Cs AMT2.2 gene was completely consistent without base mutation in the three species.Bioinformatics analysis revealed that the basic physicochemical properties,secondary structure and tertiary structure of the above proteins are relatively similar.The transmembrane helix structures all contain 11,and all are localized on the cytoplasmic membrane,which belong to membrane proteins.All of them contain Ammonium＿transp domain,and the Cs AMT2.3 protein Ammonium＿transp domain is the most prominently located.2.CsAMT2s expression was correlated with root activity and meteorological factors in different seasons.The root activity and CsAMT2s expression of different N uptake tea varieties in different seasons were measured,and the results showed that the root activity of all three varieties was higher in winter and lower in spring.The overall transcript levels of Cs AMT2.2,Cs AMT2.3,and Cs AMT2.5 were higher than those of Cs AMT2.1 and Cs AMT2.4 in the root systems of different varieties,and the correlations between CsAMT2s and root activity and meteorological factors were different.The relationship between Cs AMT2.3 and Cs AMT2.5 genes and root activity and meteorological factors was more profound.This result echoes the predicted results for the relevant cis-acting elements in the promoter region of CsAMT2s,and it can be concluded that the expression level of CsAMT2s is heavily weighted by meteorological factors.3.There was a correlation between root physiological activity and CsAMT2s transcript levels in tea tree under different fertilization treatments.The root vigor,root total N content,root NH₄⁺-N content were determined by adopting different fertilization treatments for different N uptake tea tree species.Real-time fluorescence quantification was also performed on different tissue parts of tea trees.The results showed that the fertilization treatments could improve the growth of tea trees,and the root vigor,total nitrogen content and NH₄⁺content of tea tree roots were all improved to different degrees in different nitrogen-absorbing tea tree varieties,and the relevant indexes of tea tree roots under the treatment of chemical fertilizer and organic fertilizer were the best,which provided scientific basis for guiding the fertilization of tea trees.At the same time,it was found that the nitrogen uptake performance of tea tree root system was correlated with CsAMT2s genes to different degrees,and the expression of Cs AMT2.2,Cs AMT2.3 and Cs AMT2.5 genes were positively correlated with the above indexes.It provides a new reference standard for assessing the nitrogen utilization in the root system of tea trees.4.Cs AMT2.3 gene is a membrane protein with the ability to absorb and transport NH₄⁺.The results showed that the green fluorescence of Cs AMT2.3protein was localized on the plasma membrane by means of in vitro validation of transient transformation of tobacco,defective yeast backfill and stable transformation of mutant Arabidopsis thaliana,verifying that Cs AMT2.3 protein mediates transmembrane transport of NH₄⁺as a membrane protein.Comparing the functional differences of Cs AMT2.3gene among different N uptake tea varieties,it was found that all three varieties could grow normally at external NH₄⁺concentrations less than0.5 m M,indicating that the Cs AMT2.3 gene belongs to a high affinity system.MX grew weakest in high NH₄⁺concentration environment,while when the exogenous NH₄⁺concentration was low（<0.5 m M）,HD grew better than the other two species.This indicates that there are differences in the uptake performance of different nitrogen-absorbing tea tree species for NH₄⁺,which is consistent with the characteristics of nitrogen uptake and utilization of different nitrogen-absorbing tea tree species.The biomass of transgenic lines overexpressing 35s::Cs AMT2.3was increased under low ammonium supply and showed optimal growth levels at 0.2 m M NH₄⁺concentration,again verifying that Cs AMT2.3acts as a high-affinity transporter and mediates efficient root uptake of NH₄⁺in vitro.