Identification Of Amino Acid Transporter Genes In Tea Plant[Camellia Sinensis]

Amino acids are the main components responsible for the aromar and taste of tea,and both of the content and component determine the tea quality.Given that amino acids are the main forms of nitrogen（N）assimilation products during long-distance transport process in higher plants,the content of amino acids in tea leaves is determined by the nitrogen uptake and assimilation efficiency as well as the transport efficiency and allocation pattern of amino acids in tea plant.The translocation of amino acid in tea plant occurs across membranes,including uptake by root,long-distance transportation between source-sink organs loading followed by xylem and phloem loading,and allocation among the sinks in shoots.The amino acid transporters located plasma membrane are responsible for the series of transport.So far,numerous studies have focused on the amino acids uptake and transport mechanism,and many amino acid transporters have been identified in Arabidopsis,rice,pea,poplar and other plants,however,little is known in tea plant（Camellia sinensis）.To identify candidate genes invoved in amnio acid transportation,the study has been carried out as the following aspects:（1）To identify the variations of the content and composition of free amino acids among 11 tea varieties of different tea leaves,one bud and one leaf,the second leaf,and mature leaf;（2）To determine the effects on the content of free amino acids in tea leaves（one bud and two leaves）by three various nitrogen treatments,inorganic nitrogen at normal level,equally low inorganic nitrogen and a mixture of amino acid and inorganic nitrogen using hydroponic culture;（3）To identify the candidate genes involved in nitrogen uptake and transport in response to four different nitrogen treatments,normal inorganic nitrogen（CK）,the other three treatments were resupplied with normal inorganic nitrogen（NN）,low inorganic nitrogen（LN）and amino acid nitrogen（ON,organic nitrogen）after N starvation for three days.Absorbing root tips and young shoot under different N treatments were resequenced jointly by the technologies of RNA-seq and Single Molecular Real Time（SMRT）;（4）To characterize the gene functions for the four amino acids transporter candidate genes of CsYPQ1 、CsCAT9、CsAAP3 and CsLHT8.The main results were as follows:1.Significant genetic variations of free amino acid content were observed in the 11 tea varieties with different suitability for different tea,the amino acids contents in the one bud and one leaf as well as the second leaf were significantly higher than that in the mature leaves.The amino acids contents in the second leaf were significantly higher than those of one bud and one leaf by 45.82%,16.38%,8.21% and 7.79% in the varieties of Anjibaicha,Jinguanyin,Zhenong117 and Echa10,repectively.The amino acids content in one bud and one leaf was sigificantly higher than that of the second leaf in the three cultivars of Zijuan,Wufeng212 and Fuyun6,whileno significant difference was found between a bud with one leaf and the second leaf in the varieties of Fudingdabai,Yingshuang,Zhongcha108 and Jinmudan.The content of theamine,a dominant component,in one bud and one leaf as well as the second leaf were significantly higher than that in mature leaves in the 11 varieties,nevertheless,the glutamate content was predominant in the mature leaves of Anjibaicha,Wufeng212,Zhongcha108,Jinmudan and Fudingdabai.2.The total amino acids content in 2.50 m M normal inorganic nitrogen（NH₄+&NO₃^-）treatment was significantly higher than the treatments of 0.28 m M inorganic nitrogen（NH₄+& NO₃^-）and mixture of organic nitrogen and inorganic nitrogen,containing 0.15 m M amino acid（theanine,alanine,glutamate,valine,glycine at 0.03 Mm equally）and 0.13 m M（NH₄+& NO₃^-）,simulating the status of organic garden.The seedlings grew well exposed to the three treatments.The content of theamine,argine and glutamate in the inorganic nitrogen treatment at normal level was significantly higher than those at both treatments of low inorganic nitrogen and the mixture nitrogen treatments.However,the content of alanine was highest in the mixture nitrogen treatment,significantly higher than those of other two nitrogenous treatments,and the difference of the other components was not significant among the three treatments.3.A total of 32967 unigenes were obtained through transcriptome sequencing,and24127,28869,18101 and 24466 unigenes were annotated against Nr,Swiss Prot,GO and COG databases,respectively.Totally 9592 unigenes were annotated against KEGG database and classified into 348 KEGG pathways.There were 93,7 and 75 unigenes annotated as nitrate transporters,ammonium transporters and amino acid transporters respectively.In contrast to the N-replete control,the most pronounced DEGs occurred both in shoot and root within the resupply of organic nitrogen treatment.Insterestingly,nine amino acids transporter genes,one homologue of AAP3,one homologue of LHT8,three homologue of YPQ1 and four homologue of AVT1,were uniquely differentially expressed in organic nitrogen treatment compared with the control.A large number of DEGs related to transporters,transcription factors,nitrogen assimilation enzymes,amino acid metabolismwere identified.4.Four candidate amino acid transporters of CsYPQ1,CsAAP3,CsCAT9 and CsLHT8 were cloned.Transmembrane region prediction showed that CsYPQ1,CsAAP3,CsCAT9 and CsLHT8 were transmembrane proteins.The overexpression vector of each gene was conducted and transformed into Arabidopsis wild type and mutant for functional verification.