Cloning And Functional Analyzation Of Amino Acids Transporter OsLHTl In Rice

Nitrogen is an essential element for plant growth and development.Nitrate and ammonium are the main forms of nitrogen in soil.Recent studies have shown that small amounts of amino acids in soil can also be directly utilized to support plant growth and development.In order to better solve the relationship between nitrogen uptake and transport,the research of plant amino acid transporters mainly focused on Arabidopsis than rice.OsLHT1 has been reported as a histidine transporter in yeast,but its substrate specificity and gene function remain unclear.This study will further our understanding of the substrate specificity of OsLHT1 and its roles in rice growth promotion and yield formation.1.Two mutants were obtained by CRISPR/Cas9 gene editing technology.Loss of OsLHTl function inhibited the growth of rice roots and shoots,obvious brown spots in leaves and signs of plant senescence.The yield of mutant plants,including seed setting rate,number of grain per plant seeds and 1000-grain weight of seeds,was significantly reduced.2.Substrate selectivity of OsLHT1 in Xenopus laevis oocyte was analyzed by two-electrode voltage clamp technique.It was found that OsLHT1 transported a variety of amino acids,including basic,neutral and acidic amino acids,and showed a preference for neutral and acidic amino acids,which differed from its homologue in Arabidopsis thaliana.3.QRT-PCR analysis showed that the expression of OsLHTl was lower in stem than in root,stem,leaf and young panicle.In the roots,the highest expression was detected in root tips(0-2 cm).4.Transient expression of rice protoplasts indicated that OsLHT1 was located in the plasma membrane,which was consistent with its function as an amino acid transporter.5.In order to verify the relationship between sources and plant phenotypes,the absorption experiments of amino acids,nitrates and ammonia ions in hydroponic culture were carried out.The results showed that nitrates and lysines could promote the development of plant roots,while the relationship of OsLHTl and nitrogen was complex and difficult to explain clearly.Total nitrogen measurements showed that OsLHTl might be involved in the uptake of nitrogen sources and the upward transport of amino acids.More experiments are needed to verify the specific transport and absorption.In conclusion,our results show that OsLHT1 is an amino acid transporter with wide substrate specificity and selectivity for neutral and acidic amino acids.The disruption of OsLHTl function significantly inhibits the growth and reproduction of rice.In order to further explore OsLHT1 and rice yield,amino acid transport substrates,as well as the direction of transport,provide a scientific and feasible theoretical basis.