Cloning And Functional Study Of NtAAP2 Gene In Tobacco

Amino acids are essential organic nitrogen compounds in plants.The content of a mino acid in tabacco leaves not only affact the growth of tabacco,but also affact the quality and flavor of tobacco.Amino acid transporters mediate the transport of a mino acids in plant and play animportant role in the growth and development.Amino acid transporters are divided into two major superfamilies: the amino acid,polyamine and choline transporters superfamily(APC)and the a mino acid transporter family(ATF),The AAPs family(amino acid permease)is subfamily of the ATFs,and have been extensively studied.In this study,we performed bioinformatics analysis of tobacco AAP gene family,and we successfully cloned two NtAAP2 genes: NtAAP2-1 and NtAAP2-2,and made a comprehensive analysis including their gene structure and their expression profiles.Moreover,we constructed the overexpression and RNA interference vectors of NtAAP2-1 and NtAAP2-2 and these vectors were transformed into tobacco mediated by Agrobacterium.Then,we eveluated the contents of a mino acid in tabacco leaves transgenic plants,to provide more references for revealing their roles in tobacco.The main results as follows:(1)Analysis of AAP gene family in Nicotiana tabacumHere,a total number of 15,7,and 6 full-length AAP genes have been identified in the genomes of Nicotiana tabacum,Nicotiana sylvestris,and Nicotiana tomentosiformis,respectively.We performed a multiple analyses of the AAP gene family in tobacco and here report data regarding chromosomal location,phylogenetic relationships,gene structures,predicted protein structures,and information about conserved motifs.These tobacco AAP family members shared high levels of similarity in their nucleotide and amino acid sequences.Distribution mapping exhibited that the 15 NtAAP genes were unevenly distributed among eight chromosomes,and the gene structures of the Nt AAP loci are all disrupted by introns.Phylogenetic analyses indicated that these AAP genes were clustered into four groups,and AAP genes in tobacco were mainly distributed into cluster 1 and cluster 4.(2)Cloning and bioinformatics analysis of tobacco NtAAP2-1 and NtAAP2-2 geneWe successfully cloned the coding sequence and the gene sequence of NtAAP2-1 and NtAAP2-2 from tabacco(Honghua Dajinyuan),the results of bioinformatics analysis indicate that,the full length coding sequence(CDS)of NtAAP2-1 is 1542 bp,encoding 513 amino acids whose molecular weight is 56.99 kDa and the pI is 9.52;the CDS of NtAAP2-2 is 1539 bp,encoding 512 amino acids whose molecular weight is 56.79 kDa and the pI is 9.45.Sequence identities analysis showed that the a mino acid sequences of these two NtAAP2 genes shared 69.96 % identity and the nucleotide identity reached to 71.04%.Each of the NtAAP2 proteins containd one Aa_trans domains and also have 12 transmembrane helices,which were all hydrophobic protein.Based on the multiple sequence alignment and phylogenetic analysis,NtAAP2-1 and NsyAAP2,NtAAP2-2 and NtomAAP2 were orthologous respectively.The tissue expression profile analysising of NtAAP2-1 and NtAAP2-2 indicates that both NtAAP2-1 and NtAAP2-2 sharing the same expression pattern,both of them has higher transcripts in stems and roots.(3)The creation of tobacco NtAAP2 transgenic linesTo study the function of NtAAP2-1 and NtAAP2-2,the overexpression vectors and the RNAi vector of NtAAP2-1/NtAAP2-2 were constructed and these vectors were transformed into tobacco,ultimately obtained the T1 transgenic tobacco.(4)Evaluation of free a mino acids in leaves of T1 transgenic tobaccoThe content of 18 amino acids in transgenic tobacco and wild tobacco leaves were evaluated,and results showed that the content of Asp,Asn,Glu and Gln were changed,indicating that NtAAP2-2 was involved in the transport of Asp,Asn,Glu and Gln in tobacco.