Screening And Preliminary Functional Analysis Of Transcription Factors Interacting With GA20ox1Promoter In Arabidopsis

Gibberellin is one of important endogenous hormones in plant, and involves inregulating plant growth and development. Gene expression in eukaryote is regulatedat multiple levels, especially at transcriptional level. Transcription factors interactwith promoter regulating gene expression. At present, more research has been doneabout GA20ox function in gibberellin biosynthesis pathyway, but little research hasbeen done about upstream cis-elements or promoter analysis of AtGA20ox. So weconducted, and some results of detailed research were obtained:(1) In this paper, chromosomal localization, evolution, protein motifs andcis-elments of gibberellin biosynthesis key enzymes were analyzed by bioinformaticmethods. The results exhibited that gibberellin biosynthesis key enzymes familyshowed relatively concentrated chromosomal localization, analogous introns andexons. It also revealed that the more similar the genetic evolutionary relationships is,the more conservative and the closer distribution position in motifs and cis-elementsis. The results indicated that there were a lot of conservative elements in thepromoter sequence of gibberellin biosynthesis key enzymes, including tissue andorgan-specific elements, light responsive elements, phytohormone and environmentresponsive elements. Gene chip data showed that expression of gibberellinbiosynthesis key enzymes responded to all kinds of hormone induction. AtGA20oxcould be divided into two subclasses, AtGA20ox gene had a high expression level inthe flower and silique of Arabidopsis. The promoter of AtGA20ox have more lightand phytohormone elements, also have more MYB binding elements.(2) Yeast one-hybrid system was performed screen AtGA20ox promoterinteraction proteins with Arabidopsis transcription factors library. The interactionintensity between AtGA20ox promoter and AtMYB32were measured by CPRG assaywith β-galactosidase. The results exhibited that AtGA20ox promoter interact withAtMYB32strongly.(3) The prokaryotic expression vector pCold TF/AtMYB32was successfullyconstructed. To study the best induced conditions for expression of recombinantfusion protein. The results reviewed that the optimal conditions were performed at18℃for6h with0.6mmol/L IPTG. The analysis of soluble recombinant proteinshowed that the fusion protein was present both in the supernatant and the pellet. The supernatant of pCold TF/AtMYB32was further purified by afinitychromatography and detected by Western blot, which demonstrated that high qualityrecombinant protein was obtained. It also further confirmed that the fusion proteinwas our target protein by Western blot analysis.(4)AtMYB32belongs to MYB family, and there are two conserved R2R3MYBdomains. Basic physicochemical properties and nuclear location of AtMYB32wereanalyzed by using bioinformatic methods, it was also confirmed by protoplasttransformed experiment. The result proved that AtMYB32located in the nucleus.Real-time quantitative PCR analysis demonstrated that though AtMYB32geneexpression was detected in all analyzed tissues, the relative expression in flower andsilique was significantly higher than that in other tissues.(5) By constructing35S::AtMYB32-GFP and pAtMYB32::GUS plasmid, stabletransgenic Arabidopsis lines overexpressing AtMYB32were generated throughAgrobacterium-mediated transformation.(6) Treatment wild-type and light receptors mutation Arabidopsis by continuousor transient blue, red and far red light, real-time quantitative PCR analysis reviewedthat AtMYB32gene expression was induced by light. Treatment wild-type Arabidop-sis by phytohormone or stress such as NaCl, quantitative analysis the expression ofAtMYB32, revealed AtMYB32gene expression induced by the treatments.