Cloning And Functional Analysis Of CAT Amino Acid Transporter Genes In Tea Plant(Camellia Sinesis L.)

Tea plant(Camellia sinensis(L.)O.Kuntze)is an evergreen economic perennial crops and its buds and one or two leaves below the bud are generally harvested as the best material for green tea manufacture.Amino acids are the main form of long distance transport of nitrogen in plants.The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic active source leaves to there productive sinks.In many plant species nitrogen is transported as amino acids.Amino acids,especially theanine,are important components of tea taste and health benefits.Tea plants preferably uptake ammonium nitrogen and assimilate into amino acids in roots.Amino acids represent the principal nitrogen assimilates that are transported over long distances in the vascular system to meet the metabolic nitrogen demands of tea highly diverse shoot tips and tender leaves.Therefore,the long distance transportation of amino acids in tea plants is an important process affecting the quality of tea and the nitrogen utilization in tea plants.However,the molecular mechanism controlling amino acid transport in tea plant is largely unknown.Amino acid transporters(AATs)are membrane-localized proteins that mediate the intracellular and intercellular movement of amino acids,generally from biosynthetic organs(source tissues)to utilization organs(sink tissues)via vascular tissues(xylem and phloem).Cationic amino acid transporters(CATs)are a part of the AATs family in plants,and play an important role in amino acid uptake,transport,xylem-phloem transfer and nitrogen homeostasis.However,the amino acid transport in tea plant is rarely reported.In this study,cationic amino acid transporter(CAT)family genes were cloned and preliminary physiological function are investigated,The main results are as follows:1.RACE PCR method was then used to obtain the full-length ORF of the six CsCATs.CsCATs expression was examined in various tissues,including roots,stems,leaves and flowers.CsCAT9 highly expressed in all the tested tissues and the rest of CsCATs was more specially expressed in root and stem.2.Tempo-spatial expression patterns of CsCATs and amino acid content in tea plants were also examined under exogenous amino acids and abiotic stresses.These results indicate the positive correlation between the abundance of theanine,glutamine,glutamic acid and expression of CsCATs.Our results indicated that the CsCATs had the ability to transport theanine,glutamine and glutamic acid.3.To investigate the transport functionality of CsCAT8 and CsCAT9,we cloned CsCAT8 and CsCAT9 into a pYES2 yeast expression vector and transformed into the yeast mutant strain 22?10?.Results showed that Gln,Glu,Asn,GABA and other important amino acids were the substrates of CsCAT8.The growth of 22?10?-CsCAT9 yeast strains showed that,CsCAT9 can transport asparagine,glutamic acid,glutamine,GABA,tryptophan,valine,proline and isoleucine.4.Arabidopsis thaliana mutants cat8-1 and cat8-2,cat9-1 and cat9-2 ceased to grow on L-glutamine,glutamic acid,Asparagine,GABA and theanine as the sole nitrogen source whereas wild-type plants continued to grow.Complementation strains restored to wild-type phenotype.The results demonstrate that CsCAT8 and CsCAT9 have the function to transport theanine,glutamine,glutamic acid,asparagine and GABA.It is suggested that CsCAT8 and CsCAT9 participates in the long distance transport of these amino acids from roots to leaves and seeds.Therefore,study on the expression pattern and function analysis of CsCATs will shed light on the mechanism of amino acid long distance transport mechanism in tea plants,and also provide a new way for understanding theanine synthesis and transport.At the same time,it provides important genetic resources and theoretical basis for high amino acid content and nitrogen use efficiency new varieties cultivation.