Cloning And Expression Analysis Of CsAAPs And CsLHTs Subfamily Genes In Tea Plants(Camellia Sinensis)

Tea plant?Camellia sinensis?is an important economic crop,and showed the great demand for the supply of nitrogen fertilizers.Therefore,it is critical to understand how the tea plants assimilate,transport and distribute nitrogen for increasing yield and improving tea quality.Besides the important quality component of tea,amino acid is also a nitrogen source of nucleic acids,chlorophyll,mitochondria and secondary metabolism in tea plants.It participates in various physiological processes in plants,and amino acid transporters play an extremely important role in these processes.In this study,to explore the nitrogen regulating genes responsible for the amino acid transport in tea plants,genes of AAPs and LHTs subfamilies were cloned and bioinformatics characteristics were analyzed.Meanwhile the expression patterns of those genes were studied under different cultivars,tissue parts,nitrogen levels and nitrogen treatment time.The aim is to find out the important amino acid transporter genes regulated by nitrogen level in tea plant and provide the research basis for further study on the amino acid transport regulations and the selection and breeding of nitrogen-efficient cultivars in tea plant.The main conclusions are as follows:1.According to the tea plant transcriptome information,five CsAAPs genes,named CsAAP3,CsAAP4,CsAAP6,CsAAP7 and CsAAP8,were cloned and submitted to the NCBI database.The phylogenetic tree showed that the five CsAAPs were clustered into three subgroups.The amino acid sequence alignment displayed that the five CsAAPs sequences have high homology.CsAAPs contain9-10 transmembrane regions and 16-18 AAP conserved motifs.Expression levels from different tissues showed that these five CsAAPs were extensively transcribed in these four tea cutting seedlings.The expression of CsAAP3 was higher in stem and mature leaf;the main expression tissue of CsAAP4 was mature leaves;the expression of CsAAP6 was higher in young leaves and mature leaves than roots and stems;CsAAP7 was mainly expressed in young leaves and stems;CsAAP8 was mainly express in roots and stems.2.Based on the transcriptome data of tea plants,five CsLHTs genes which were CsLHT1,CsLHT2,CsLHT6,CsLHT8.1 and CsLHT8.2,were obtained and four CsLHTs genes were successfully cloned except for CsLHT2.The results of amino acid sequence alignment and phylogenetic tree construction showed that the sequence similarity of CsLHT8.1 and CsLHT8.2 was 53.9%and clustered in the same branch,indicating that they were related closely.The predicted results of conserved domain showed that the three-dimensional structure of subfamily is similar to SLC5-6-like_sbd subfamily.Moreover,CsLHT1 and CsLHT2 contain the domain of Aa_trans?PF01490?.The tissue expression results showed that four CsLHTs were expressed in all vegetative tissues.CsLHT6 is mainly expressed in roots and stems;the expression of CsLHT8.2 gets the maximum in stems which is over four times in comparison with other tissues;CsLHT1 and CsLHT8.1 are expressed in mature leaves and roots.3.The results of real-time PCR showed that nine genes of two subfamilies,CsAAPs and CsLHTs,showed different variation patterns in three nitrogen levels and three cultivars in tea plants.The results showed that the subfamily gene expression of amino acid transporter was affected by both nitrogen level and genotype.The genotype of variety had great influence on CsAAP3 gene expression,and the expression levels of CsAAP3 and CsAAP8 were decreased with the increase of nitrogen levels in stem of ZC302 which was identified as a high nitrogen efficient cultivar.CsLHT8.1 has an obvious response to nitrogen treatment,and the response model to nitrogen varies with different cultivars.Especially,after72h treatments of 0.2mmol·L^-1 and 10mmol·L^-1 NH₄NO₃,the CsLHT8.1 expressions were significantly up-regulated in the root of‘Zhongcha 302'?ZC302?.These results implied that CsLHT8.1 might participate in the transport of amino acids from root to aerial parts in tea plants.