北美鹅掌楸LtAGO1 基因的克隆、表达及其启动子分析

GO1 plays an important role in the differentiation of leaf primordia, originating from the theperipheral region of Shoot Apical Meristem(SAM). To study the morphogenesis mechanism of leaf primordiumdifferentiation, we cloned 2 001bp upstream region of LtAGO1 CDS as the promoter by RT-PCR and RACEtechnology on the basis of previous cloned LtAGO1 gene sequence, and predicted its function. Real-time PCR wasused to investigate expression pattern in Liriodendron L. We obtained the transgenic Arabidopsis thaliana ofProAGO1::GUS by resistance screening and DNA dentification, and then monitored phenotype and GUShistochemical staining. The results were as follows: (1) The LtAGO1 gene included an open reading frame for3300 bp , encoded 1 100 amino acid , the molecular weight was 122.14 kD and theoretical isoelectric point was 9.36. (2)Amino acid sequence analysis showed that it consisted of Gly-rich-AGO1 and Piwi conserved domains ofAGO family. Phylogenetic trees revealed that LtAGO1 was closed to Cinnamomum micranthum RWR84608.1in evolutional relationship. (3)The specific tissues expression analysis demonstrated that the expression order wasthat stamen>floral bud>petal>calyx>leaf>pistil>leaf bud>stem among tissues, and the expression order was thatleaf bud sprouting stage >young leaf stage>senescence stage >mature stage among stages. It was highly expressedin the leaf margin of Liriodendron L, and LtAGO1 gene expression in leaf tooth sinus was higher than in leaf toothtip of Liriodendron tulipifera. (4)The transgenic strains leaf polarity of the middle and basal apical axis wasabsent with serrated leaf margin and double petal flower. It was found that GUS staining was stably detected at thetip of leaf bud of transgenic seedlings , the higher GUS activity was observed at newly differentiated petioles.LtAGO1 promoter drove GUS gene to accumulate specifically in the vascular bundle of Arabidopsis thaliana leaf ,flower, pod and stem, and GUS activity intensity order was that leaf tip bud> flower>vascular bundle amongtissues, which was accordance with the Real-time PCR results in Liriodendron tulipifera. Therefore, the resultsalso showed that LtAGO1 gene was predominantly expressed in apical meristem and regulated by variouspathways during the development of leaf and flower. It will provide a foundation for further functional research ofAGO1 protein and regulation mechanism of leaf shape development.