Amino Acid Transporter And Protocorm Formation Of Dendrodium Officinale Kimura Et Migo

Dendrobium officinale Kimura et Migo belongs to the genus Dendrobium,Orchidaceae,Monocotyledons,Angiosperm,and is a perennial epiphytic herb.According to the morphological characteristics of protocorm,the seed germination process can be divided into seven stages: seed embryo activation stage(P1),protocorm formation stage(P2),primary meristem formation stage(P3),apical meristem formation stage(P4),ellipsoid protocorm stage(P5),leaf primordial vascular system formation(P6)and root meristem formation formation(P7).Amino acid transporter is a kind of membrane protein mediating transmembrane transport of amino acids in advanced plant.It mainly includes amino acid-polyamine-choline transporter family(APC)and amino acid/auxin osmotic enzyme family(ATF).APC family contains two subfamilies,including cationic amino acid transporters(CATs),L-type amino acid transporters(LATs);ATF family includes amino acid permease(AAPs),lysine histidine-like transporters,(LHTs),proline transporters(ProTs),auxin-resistant(AUXs),?-aminobutyric acid transporters(GATs),aromatic and neutral amino acid transporters(ANTs).In this study,D.officinale was used as a material to explore the structure and function of amino acid transporters through bioinformatics analysis,transcriptome data analysis and real-time fluorescence quantification.The main results are as follows:(1)In this study,22 DoAATs were identified from the whole genome of D.officinale by Blast et al.,including 5 DoCAT,6 DoAAP,3 DoLHT,1 DoProT,2 DoAUX,1 DoGAT,1 DoANT,3 DoLAT.Both subfamilies in the APC family have one or more AA permease domains.In addition,CATs have an AA permease C characteristic domain.Each subfamily in the ATF family contains an AA trans domain,among them,LHTs also contain a Trp-Tyr-perm specific domain.Each of the DoAATs contains a transmembrane domain and has no signal peptide,which is a hydrophobic protein.In the gene structure,DoAATs have hormone response elements,stress response elements,tissue expression elements,and binding site elements,metabolic regulatory elements and physiological action components.In addition,the results of the phylogenetic tree indicated that the amino acid transporter of D.officinale was most closely related to the Phalaenopsis equestris,which is consistent with the characteristics of the same orchids.(2)The second and third generation transcriptomes were sequenced by the high-throughput sequencing technique for the P1-P5 protocorm of the D.officinale.Sequencing analysis showed that 19 DoAATs were expressed during this period,including 5 DoCAT,3 DoLAT,4 DoAAP,2 DoLHT,1 DoProT,1 DoAUX,1 DoGAT,and 2 DoANT-like.The results of structural analysis indicated that the superfamily member sequence has alternative splicing,SSR and other structures,and the variable splicing structure makes the gene different splicing isoforms.GO analysis indicates that members of the family are components of various membrane structures and all of them are involved in transmembrane transport,DoCAT mainly mediates the transport of lysine,arginine and glutamate,DoAAP family is mainly involved in the transport of acidic and neutral amino acids,DoProT2 is involved in the transport of proline and DoGAT1 is involved in ?-aminobutyric acid transport.In addition,this family of proteins is also associated with certain physiological processes,such as the process of toxin decomposition,regulation of hypersensitivity reactions,and negative regulation of programmed cell death.DoAUX is involved in the plant hormone signal transduction pathway and is involved in the growth and development of plants affected by hormones.The results of differential gene expression indicated that DoAAP2,DoAAP4 and DoLHT1 were highly expressed in P1-P5 phase,and DoCAT1,DoLHT8 and DoGAT1 were low or not expressed in P3-P5.(3)In this paper,the relative expression levels of this subfamily gene in various stages of D.officinale embryo development,tissues and under cold and drought stress were quantified by real-time PCR.The results showed that the DoAAP gene was expressed in different stages of D.officinale protocorm development,but all genes were up-regulated and down-regulated during P1-P6,and the expression increased during P7.Under the cold and drought stress,the expression pattern is the same,DoAAP2 and DoAAP3 belong to the positive feedback response.DoAAP4,DoAAP7 and DoAAP8 belong to negative feedback response.the expression of DoAAP6 is irregular.The expression of DoAAP6 is lower than that of the control sample and fluctuates up and down.When drought stress,the expression of DoAAP6 is higher than that of the control sample only at 72 h.The expression level is lower in other time periods.In conclusion,amino acid transporters can mediate the transmembrane transport of amino acids during the P1-P5 stage of seed development of D.officinale.The amino acids transported by different subfamilies are different.They provide raw materials for the formation of various proteins and play an important role in the formation of primordial meristem and apical meristem during seed development.Among them,the expression pattern of DoAAP showed that the members of this subfamily had higher expression at P7 stage,which indicated that they had the strongest transporting activity of amino acids during the formation of root meristem,indicating that they played a significant role in root formation and growth.In addition,DoAAT participates in the process of toxin decomposition,the regulation of hypersensitivity and the negative regulation of programmed cell death,which indicates that DoAAT family provides guarantee for the normal embryonic development of Dendrobium officinale Kimura et Migo.