Study On Key Genes In Gibberellin Biosynthesis And Signaling Transduction During Early Peanut Pod Development

Gibberellins （GAs） regulate plant growth and development in many aspects.GAs promote the transition from meristem cell to shoot development, from thejuvenile leaf to the adult leaf development, from vegetative growth to flowering, andaffect the development of the flower. Proper adjustment of these growth anddevelopment process is essential for the survival of the plants and production of crops.Fruit development could directly affect seed yield, quality and disease resistance andunderstanding the mechanisms of fruit developmental is of great importance.Understanding the regulation roles of gibberellins on peanut pod development couldbe an effective way to clarify the mechanism of peanut pod development.GA₂0oxidase and GA₃oxidase plays a key role in gibberellins biosynthesis andgenerating biological active gibberellins. GA₂oxidase is a key enzyme for biologicalactive gibberellin inactivation. GA₂0oxidase, encoded by a multigene family, is therate-limiting enzyme of gibberellin biosynthesis in many plants. Gibberellin3-beta-oxidase （GA₃-ox） is also named as gibberellin3-beta-hydroxylase catalysesGA₂0to GA1and GA9to GA4, respectively. GA₃-ox is also a rate-limiting enzyme ofgibberellin biosynthesis. GID1is the gibberellin receptor and DELLA is a negativeregulator of gibberellin signaling pathway. DELLA directly or indirectly inhibits thetranscription of gibberellin response genes when the bioactive gibberellins are absentor when the concentration of bioactive gibberellins is low. Bioactive gibberellinscould bind to GID1, and leading to the conformational change of GID1which couldinteraction with DELLA proteins. The interaction between GID1and DELLApromotes DELLA ubiquitination and leading to DELLA degradation through theubiqitin proteasome pathway. The bioactive gibberellins regulate plant growth anddevelopment by removal of DELLA repression and leading to the activation of gibberellin responsive genes. In this study, we cloned the key genes in gibberellinbiosynthesis and signaling pathway, and verified their function. The results laid thefoundation for studying the molecular mechanism of peanut pod development.The main results of this experiment are as follows:（1） Gene cloning: The peanut transcriptome data was analyzed and thecorresponding Unigenes were obtained and were used to design5’RACE and3’RACEprimers. Full-length ORF of AhGA₂-ox （1014bp）, AhGA₃-ox （1095bp）, AhGA₂0-ox（1128bp）, AhDELLA（1821bp） and AhGID1（1035bp） was amplified from peanut.（2） Vector construction and tobacco transformation: AhGA₂-ox, AhGA₃-ox,AhGA₂0-Ox and AhGID1overexpression vectors were constructed and were namedas pCAMBIA₂300-AhGA₂-ox, pCAMBIA₂300-AhGA₃ox, pCAMBIA₂300-AhGA₂0-ox and pCAMBIA₂300-AhGID1. Agrobacterium-mediated transformmethod was used for tobacco transformation. After co-culture, antibiotic selection androot generation, transgenic lines expressing AhGA₂-ox, AhGA₃-ox, AhGA₂0-Ox andAhGID1were obtained.（3） Bimolecular fluorescence complementation （BIFC） experiments and yeasttwo-hybrid verification of DELLA and GID1interaction: BIFC vectors wereconstructed and named pSPYCE-AhDELLA, pSPYNE-AhGID1. Yeast two hybridvectors were constructed and named pGBKT7-AhGID1, pGADT7-AhDELLA. Theinteraction of these two proteins was observed in both BIFC and Yeast two hybridexperiments.（4） The expression analysis of AhGA₂-ox, AhGA₃-ox, AhGA₂0-ox, AhDELLAand AhGID1: Gene specific primers were designed and were used to analyse theexpression of these genes in different tissues by qRT-PCR. The results showed that theexpression patterns varied in different tisssues and organs.