| Submergence stress does severe harm to rice production. In the long-term rice evolution, it has evolved a mechanism and some strategies to adapt to submerged stress. Furthering study the tolerance mechanism from the molecular level, which can offer a theory for other plants, and other plants also can improve their genes to resistance the submerged stress and increase their crop production.Here we chose artificial miRNA-based strategy to knock down gene expression in rice, we use endogenous rice osa-MIR528precursor as the backbone to design amiRNA. This study is aimed at assessing the functions of synthase of the important hormone by artificial microRNA. This study is aimed at assessing the functions of submerged stress promotes rapid growth of rice coleoptile.Current methods for constructing amiRNA expression vectors is costly and suffers from mutation problems. In this study, we developed a efficient, simple, low-cost and high-throughput approaches to generate amiRNA expression vectors in rice, we constructed amiRNA expression vectors based on one-step PCR process and mating-assisted genetically integrated cloning (MAGIC). The main study results are as follows:(1) The gene of OsGA20oxl and OsGA20ox2specific amiRNA expression vectors pmiR528-OX1-1, pmiR528-OX1-2and pmiR528-OX2-1, pmiR528-OX2-2were constructed based on one-step PCR and MAGIC;(2) The amiRNA expression vectors was transformed into rice callus by Agrobacterium tumefaciens-mediated rice transformation;(3) High frequency of callus induction and plant regeneration of recipients are very important for genetic transformation. Using the high efficient and stable of the rice tissue-cultured regeneration system established in my study, the positive callus were differentiated into plants.The PCR analysis showed that the ratio of positive transgenic lines was60%;(4) The results of quantitative RT-PCR suggested that8’-hydroxylase transcript level was reduced by68～81%in the transgenic lines of pmiR528-ABA4expression vectors. |
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