Expression Analysis Of MdASN Family And Functional Research Of MdASN2 In Malus

Apple is one of the four major fruits in the world.The planting area and yield of apple in China account for more than 50% of the world total.Nitrogen supply capacity was closely related to yield,quality and resistance,and storage nitrogen was important for early spring growth,blooming,fruit setting and early fruit development of apples.Asparagine is the main carrier for source-to-sink nitrogen reuse during apple leaf senescence,and asparagine synthetase is the key enzyme for asparagine synthesis.In this paper,we applied bioinformatics method to identify MdASNs in apple,studied the expression pattern of MdASNs gene in the senescence process of ’Fuji’ apple induced by exogenous abscisic acid,analyzed the role of MdASN2 in the synthesis of asparagine and nitrogen reuse in apple leaves.This provides a theoretical basis for improving the utilization rate of nitrogen in apple.The main research results are as follows:1.By bioinformatics analysis,four MdASN genes were identified in the apple genome,which were distributed on four chromosomes,respectively.The coding sequence length was 789～1761 bp.Th amino acid length was 262～586 aa,all of which have conserved GATase＿7 and ASN domains.Expression analysis showed that MdASNs were highly expressed in roots and stems,and MdASN2 was highly expressed in mature leaves and induced by abscisic acid.2.The effects of 500 μmol/L abscisic acid(ABA)on senescence and nitrogen metabolism were studied in two-year-old ‘Fuji’ potted plants by RNA-Seq.The results showed that exogenous abscisic acid decreased the photosynthetic rate and maximum photochemical efficiency of photosystem Ⅱ(Fv/Fm),caused leaf senescence and reduced the total nitrogen content in leaves.To analyze the difference of transcription level in apple leaves after ABA treatment for 6 and 12 days.20 ABA signaling pathway genes(PYR/PYL,PP2 C,SnRK2 and ABF),18 nitrogen metabolisation-related genes(GLN1,GDH and ASN)and 217 transcription factor(ERF,WRKY,NAC,bHLH,C2H2,MYB and bZIP)were differentially expressed.3.The MdASN2 gene was cloned from the leaves of ‘Fuji’ apple.The MdASN2 gene was located in the chromosome 3 of apple,with a total length of 1761 bp and encoding 586 amino acids.The expression of MdASN2 in mature leaves was significantly higher than that in young leaves.The subcellular localization of MdASN2 was in cytoplasm.Transient transformation of Orin callus showed that overexpression of MdASN2 increased the activity of asparagine synthetase and the content of asparagine,which proved that MdASN2 was involved in the synthesis process of asparagine.At the same time,the promoter(1617 bp)of the MdASN2 was cloned from Malus domestica and the cil-acting elements of the promoter were analyzed.By using yeast one-hybrid screening system,we obtained two candidate transcription factors MdbZIP11 and MdbZIP46 for regulating the MdASN2 promoter.