The Research And Application Of NACs In The Process Of Pear Seed Development And Dormancy Release

Seeds are the beginning of most plant life cycles.Seeds of perennial woody plants usually ensure that seeds germinate in suitable time and environment by dormancy.Pear seeds are dormant.At present,the molecular mechanism of dormancy in pear seeds is still unclear.As plant-specific transcription factor,NAC is involved in many physiological processes including seed development,secondary wall growth,flower and fruit maturation.However,its role in seed dormancy has not been reported.In this dissertation,the seeds of Pyrus calleryana Decne were used as research materials.The changes of sugar substances(glucose,fructose,sucrose and starch)and hormones(GA and ABA)during the development of Pyrus calleryana Decne seeds,and the changes of reactive oxygen species,its metabolic related enzymes and hormones(GA and ABA)during dormancy release by HPLC,DNS colorimetry,enzyme linked immunoassay and ultraviolet spectrophotometer,and the antisense oligonucleotide technology was used to research the changes of NAC.The main results were as follows:1.The structural characteristics of nine NAC genes were determined.Taking the target gene of miRANA164-nine NAC transcription factors involved in dormancy release of Pyrus calleryana Decne seeds as the object,bioinformatics method was used to find that the protein encoded by nine NAC genes contained 290-362 amino acids,the molecular weight of the protein was about 32604.7-40415.6 Da,and the isoelectric point was 6.1775-8.3422.PpNAC5,PpNAC134,PpNAC86,PpNAC42,PpNAC121,PpNAC34 and PpNAC138 were basic amino acids,while PpNAC65 and PpNAC145 were acidic amino acids.The values of protein hydrophobicity showed that only PpNAC86 was amphoteric amino acid and the other eight NAC were polar amino acids.Systematic classification and functional predictions indicated that nine NAC proteins belong to class ?,PpNAC34 and PpNAC138 were classified as NAC1 subgroup,which mediated hormone signaling pathways;the remaining NACs were classified as NAM subgroup,in which PpNAC5,PpNAC134 and PpNAC86 may be related to the embryonic development and germination of seeds.2.Determine the content changes of sugar substances and hormones,and the expression of NAC genes in the development of Pyrus calleryana Decne seeds.During the development of the whole Pyrus calleryana Decne seed,the content of glucose decreased first and then remained stable,while the content of sucrose increased first and then stabilized,and the content of starch was stabilized at an early stage and increased significantly later.The content of ABA increased gradually,while the trend of GA content was reduction—stabilization—reduction.Based on the expression of NAC,the down-regulation trend of PpNAC65 was similar to that of GA.The up-regulation trend of PpNAC34 and PpNAC138 was similar to that of ABA,suggesting that hormone metabolism may be closely related to PpNAC34,PpNAC65 and PpNAC1383.The expression of NAC gene during the dormancy release of Pyrus calleryana Decne seeds was determined,and the role of PpNAC5,PpNAC65 and PpNAC138 in the dormancy release of Pyrus calleryana Decne seeds were further confirmed.Antisense oligonucleotide technology was used to inhibit the expression of NAC gene.The research showed that the seeds after inhibition treatment germinated earlier than the normal seeds.When the expression of PpNAC5 was inhibited,the content of GA,ROS and the relative expression levels of synthetic genes were significantly increased.When the expression of PpNAC65 was inhibited,the content of GA and the relative expression of synthetic genes increased significantly.When the expression of PpNAC138 was inhibited,the content of ABA and the relative expression levels of synthetic genes were significantly decreased,while the content of GA and the relative expression levels of synthetic genes were increased significantly.The results showed that PpNAC5 was involved in the synthesis pathways of GA and ROS,PpNAC65 was involved in the synthesis pathway of GA,and PpNAC138 was involved in the synthesis pathways of GA and ABA.Through the above studies,the results provided preliminary evidence that NAC was involved in the physiological processes about development and dormancy release of Pyrus calleryana Decne seeds.The function of NAC gene in the development and dormancy of pear seeds was further enriched,laying a foundation for further exploring the function of NAC,and provided theoretical basis for the establishment of fast breaking seed dormancy in production and cultivation of high-quality and efficient pear seedlings.