| Maize (Zea may) is the largest cultivated crop in the world, and it is not only an important food crop, but also an essential economic crop. It is also widely used in food, feed and other industries, playing a very important role in the development of national economy. Starch is the main storage material for maize, so it has an effect on the quality and yield of maize. During the grain filling period, activities of key enzymes of starch synthesis and accumulation quantity and rate of starch were significant positive correlation in the endosperm of grain, therefore, to understand the genes expression of key enzymes of starch synthesis is an important way to analyze starch synthesis, however, the genes expression of key enzymes of starch synthesis in the molecular mechanism is still not clear.ABA is a significant plant hormone, which plays a vital role in plant stress resistance, seed development and grain filling. ABA promotes seed dormancy, the change of stomatal conductance, the delay of growth, the accumulation of osmotic adjustment substances and the expression of genes involved in plant development and stress resistance. During the period of seed development, endogenous ABA plays an important role as a positive regulator. Our previous studies also showed that ABA could increase many genes expression involved in starch synthesis, but the regulation mechanism is still obscure.This study took Maize Inbred Line B73 as materials, whose endosperm (10 DAP, days after pollination) was treated with ABA, The RNA was isolated from samples and control, and sequenced by RNA-sequence technology. This thesis focuses on the regulation mechanism of candidate gene ZmZFP1. The results as follows:1. ZmZFP1 gene was cloned and its advanced structure was predicted. The results showed that ZmZFP1 contained the typical structure of zinc finger protein including secondary structure, tertiary structure and domain. The results of phylogenetic tree and conserved motif showed that ZmZFP1 contained a conserved domain. ABA relative cis-acting elements in the promoter of ZmZFPl was online analyzed using Plant-CARE software. The results showed that the ZmZFPl promoter contained a cis-acting element for the induction of ABA.2. Expression of ZmZFPl was detected in endosperm by real-time quantitative PCR analysis, and the results showed that it was constant with sequencing analysis results. The expression of ZmZFPl gene was induced up-regulation expression by ABA.3. Construction of ZmZFPl and promoter +Luc expression vector, gene gun bombarded 10 DAP maize endosperm, and Lus and Gus activities were measured, the results indicated that ZmZFPl transcription factor could improve the activity of promoter Sh2, Btl and Wx, and it is indicated that the ZmZFP1 could promote theses gene expression.4. Analysis of the transcriptional activation activity of ZmZFP1 that was taken the yeast activation experiment. The results displayed that PGBDKT7-ZmZFPl was able to grow on the defect type culture medium of SD/-Trp/-His/X-agal, indicated that ZmZFP1 has transcriptional activation activity.5. To construct the fusion expression vector of ZmZFPl and green fluorescent protein GFP, subcellular localization of ZmZFP1 protein was analyzed in onion epidermal cells by gene gun bombardment. The results showed that ZmZFP1 protein was localized in the nucleus. It was suggested that ZmZFPl may be involved in the expression of transcriptional level.6. The ZmZFP1 combined with promoters of Sh2, Btl and Wx genes were analyzed by yeast one hybrid experiment. The results showed that ZmZFP1 was combined with promoter Sh2 and promoter Wx, respectively. |
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