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Cloning and Characterization of Photoperiod Sensitive Gene ZmELF4 in Maize

Author: ZhangShaoFang
Tutor: ChenYanHui
School: Henan Agricultural University
Course: Crop Genetics and Breeding
Keywords: maize photoperiod sensitivity EARLY FLOWERING 4 real-time PCR Transgene subcellular localization
CLC: S513
Type: Master's thesis
Year: 2011
Downloads: 15
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Tropical and subtropical maize germplasm contain abundant hereditary variation,which can broaden the genetic base of Chinese maize germplasm by improving and using of these germplasm. However,photoperiod sensitivity severely limits the use of tropical and subtropical germplasm in temperate zone.In the result,isolating,cloning, analyzing the function of photoperiod-sensitive genes and revealing photoperiod-sensitive from the molecular level, passiving photoperiod-sensitive is an important way to research the molecular mechanism of maize’s photoperiod-sensitivity. We cloned the ZmELF4 gene by the method of orthologus gene cloning in a photoperiod-sensitive tropical maize (Zea mays L.) inbred line CML288 and a photoperiod-insensitive temperate inbred line HuangZao4,which was the cognate of ELF4 gene(Arabidopsis) and ELF4 gene(rice). Then we studied the circadian rhythm expression pattern of ZmELF4 by fluorescence quantitative RT-PCR, analyzed the expression pattern of ZmELF4 in shoot apical meristems (SAM) and leaves,and researched the relationship among ZmELF4, ZmCOL, and ZmCCA1,in order to investigate the function and mechanism of ZmELF4 during the photoperiod induction.We constructed the fusions vectors and the over expression vectors of ZmELF4, and carried subcellular localization by the onion epidermis transient expression. Then we constructed the over expression vectors of ZmELF4 transformed it into the Arabidopsis with Floral Dip method. The main results are as follows:1.We had cloned the maize ZmELF4 gene by orthologus gene cloning in the first,which GenBank accession number was HQ009862. The gene has no intron, but there was a insertion sequence of 169bp in the 5 ’untranslated region of DNA sequences. However,this sequence is absent in the cDNA sequences.The cDNA sequences which we had had were about 680bp,contained a predicated 432bp open reading frame, which could encode a peptide with 144 amino acids. By phylogenetic analysis, we found ZmELF4 and OsELF4 in the same evolutionary branch, their relationship were closest. The predicated protein that especially containing DUF1313 unknown domain, and predicated it may be homologue of ELF4/OsELF4.2.We used fluorescence quantitative RT-PCR to research the circadian rhythm expression pattern of ZmELF4 under long day and short day conditions in CML288 and HuangZao4, analysesed the expression pattern of ZmELF4 in shoot apical meristems (SAM) and leaves, and researched the relationship among ZmCCA1 and ZmCOL. The results showed that ZmELF4 gene exhibited diurnal expression patterns both in SAM and leaf under long day and short day conditions in CML288 and HuangZao4, but this rhythm is unchanged when we afford with continuous light,the differerce is only the differernt expression.The expression peak of ZmELF4 gene is unanimous under corresponding long day and short day conditions in CML288 and HuangZao4, but the overall expression is different. In different development stages of maize, ZmELF4 gene expressed in different tissues,but expressed significant differences in the two materials.Moreover,compared the expression pattern of ZmELF4, ZmCCA1 and ZmCOL, their peak of expression all in the sensitive photoperiodic induction of flowering period, they may interact through some pathway to complete the process of floral induction of maize.3. We have constructed GFP Fusion expression vector pGIT-ZmELF4-GFP and high over expression vector pBI-ZmELF4,a transient expression assay in onion bulb epidermal cells by gene gun bombardment, the result showed that ZmELF4 can localize GFP to the nucleus of the cells. And we constructed the over expression vectors of ZmELF4, transforming it into the Arabidopsis with Floral Dip method,obtained 6 positive transformation seedlings, compared with wild Arabidopsis, the flowering time delayed an average of 25 days and more than 10 leaf numbers.

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