Cloning And Genetic Transformation Of PnAlaAT Gene In Populus Simonii×Populus Nigra

Populus simoniixPopulus nigra is a kind of populus arbor belongs to Salicaceae which has good characteristic of resist to drought,cold and barren.It is the generalized forest planting in the north area.Alanine aminotransferase(AlaAT)is pyridoxal-5□-phosphate-dependent(PLP)transferase that plays important connection function between carbon and nitrogen metabolism in plant.In this study,we characterized and identified 4 genes of AlaAT gene family in poplar genome datebase by bioinformatics method.And then,Populus simonii×Populus nigra was used to clone the coding sequence of PnAZaAT1-4 genes.Bioinformatics of the CDS,amino acid sequence of the protein were analyzed.The expression pattern of these genes in different organs,nitrogen treatment,circadian rhythm and light treatment was analyzed by real-time fluorescence quantitative PCR.The plant expression vector of pROK Ⅱ-PnAlaAT3 was constructed and transformed into Populus simonii × Populus nigra via Agrobacterium infestation.Seventeen transgenic Populus simonii×Populus nigra lines were acquired.The major results are as follows:(1)4 genes of AlaAT gene family were identified in poplar genome database by bioinformatics method.The results of phylogenetic tree indicated that four alanine aminotransferase homologues belonged to two subgroups.Subgroup A contained PtAlaAT3 and PtAlaAT4,encoding AlaAT enzymes.Subgroup B contained PtAlaAT1 and PtAlaAT2,encoding glutamate:glyoxylate aminotransferase(GGAT).(2)Four PnAlaAT genes(PnAlaAT1,PnAldAT2,PnAlaAT3 and PnAlaAT4)belonged to AlaAT gene family were obtained successfully from Populus simonii×Populus nigra by RT-PCR method.The total length of nucleotide sequence in each of the PnAlaAT1-4 cDNA was 1446 bp,encoding 481 amino acids residues.GenBank database accession numbers of PnAlaATs were KT768060,KT768061,KT768062 and KT768064 respectively.Homologous sequence alignment results showed that the nucleotide sequences consistency between the four PnAlaATs genes and corresponding PtAlaATs of Populus trichocarpa were 99%;the consistency with the amino acids sequences of protein were 100%.(3)Real-time PCR results revealed that PnAlaATl and PnAlaAT2 were expressed predominantly in leaves under normal conditions.When treated with nitrogen,expression of PnAlaATl and PnAlaAT2 were induced by exogenous nitrogen in leaves,but inhibited by exogenous nitrogen in roots.The transcript abundances of PnAlaATl and PnAlaAT2 exhibited a regular fluctuation with the diurnal changes in leaves.PnAlaAT3 and PnAlaAT4 expressed in roots,stems and leaves under normal conditions.Expressions of PnAlaAT3 and PnAldAT4 were induced by exogenous nitrogen in roots,stems and leaves when treated with nitrogen.The expression of PnAlaAT3 was induced by 10 mmol/L NH4+ significantly in roots.Additionally,when glutamine synthetase was inhibited by MSX in roots of Populus simonii×Populus nigra,expression of PnAlaAT3 gene was induced by glutamine.(4)The plant expression vector pROKⅡ-PnAlaAT3 was constructed successfully.After transformed into Populus simonii×Popwulu nigra via Agrobacterium infestation,seventeen transgenic lines were acquired.PCR and real-time fluorescence quantitative PCR tested results confirmed that PnAlaAT3 gene have been integrated into the genome of Populus simonii×Populus nigra successfully and expressed.The present research would lay the experimental foundation for further illuminating the role of alanine aminotransferase of woody crops in nitrogen absorption and utilization process.