Study For Molecular Mechanism Of ABA Metabolism Related Genes On Regulating Dormancy In Peach

An experiment was performed in state key laboratory of crop biology of Shandong Agricultural University and Shandong Institute of pomology(Tai'an), with ‘Zhongyoutao 4' peach as experiment material. Some genes relating to ABA metabolism were found through homologous alignment. qRT-PCR was used to detect the expression patterns of the genes relating to ABA metabolism in the period of seed and bud dormancy, and then the similarities and differences between them were analyzed. In addition, three members of CYP707 A members were cloned in flower buds, and then the proteins of them were successfully expressed in prokaryotic expression system. We also obtained transgenic Arabidopsis strains which over-expressed the PpCYP707A1, PpCYP707A2 and PpCYP707A3 gene. The main results were as follows:(1) The protein of ABA metabolism related genes in Arabidopsis were obtained in Phytozome database, and then blasted peach genome, deleted redundant sequences. Finally some genes relating to ABA metabolism were found in peach. Results are as follows: PpCYP707A1(ppa005059m), PpCYP707A2(ppa005020m), and PpCYP707A3(ppa005226m) were chosen as candidate genes of the CYP707 A gene family. PpNCED1(ppa002804m), PpNCED2(ppa002314m), PpNCED3(ppa014647m) and PpNCED4(ppa006109m) belonged to NCED gene family. PpAAO1(ppa000263m) was the candidate gene of the AAO gene family. ZEP and SDR1 are single genes belonging to the LOS gene family and SDR gene family, respectively. After analysis of these two gene families, the genetic relationship of ppa002248 m and ppa009814 m was quite close to ZEP and ABA2; therefore ppa002248 m and ppa009814 m were chosen as PpZEP and PpSDR1 candidate peach genes.(2) qRT-PCR was used to study the expression patterns of ABA related genes in the period of bud dormancy. Data showed that the expression patterns of ABA related genes were similar between vegetative bud dormancy and flower bud dormancy, which illustrated that ABA regulating flower bud dormancy and vegetative bud dormancy maybe have the similar molecular mechanism. NCED gene family played an important role in the period of paradormancy and endodoramncy. PpCYP707A2 and PpCYP707A3 were important genes playing critical roles in the stage of bud dormancy release.(3) qRT-PCR was used to study the expression patterns of ABA related genes to study the differences and similarities between seed dormancy and bud dormancy. Data showed that PpNCED1 and PpNCED2 increased in the stage of seed development, expression of PpCYP707A2 and PpCYP707A3 were the highest during the stratification of seeds under 4°C, which was similar to bud dormancy.(4) The promoter of the members of PpCYP707 A gene family through plantCARE was analyzed. Data showed that all of them contained ABRE-motif and GARE-motif. But in flower buds, only PpCYP707A2 was induced by GA3 and ABA.(5) A sense expression vector PBI121-PpCYP707A1, PBI121-PpCYP707A2 and PBI121-PpCYP707A3 were constructed, and transferred into Arabidopsis. PCR identification showed that PpCYP707A1, PpCYP707A2 and PpCYP707A3 had been expressed successfully in Arabidopsis.(6) The recombinant plasmids PGST-PpCYP707A1 and PGST-PpCYP707A2 were expressed in a prokaryotic expression system after its transformation into E. coli BL21. Finally, the fused PpCP707A1 and PpCYP707A2 protein by recombinant prokaryotic expression were successfully obtained and purified, and SDS-PAGE showed that the expressed proteins consistent with the size of expected protein.