| Fungus diseases have long been a great threaten to the production and quality of maize crop. Great efforts have been done to deal with these diseases, but few cultivars are efficient enough to maintain their disease-resistant ability for a long time because of the quick variation of pathogens or the friability of disease resistance of the cultivars. Traditional breeding techniques are both time and labor consuming, which makes it hard for them to be employed nowadays when elite corn cultivars resistant to fungus are in great needs. What further limited the successful application of traditional breeding technique in maize breeding is that resistance genes are scarce in maize crop. So gene engineering technique as an efficient breeding method applying to maize breeding will definitely accelerate the course of maize breeding as well as enrich the resistance resources to fungi pathogens because this technique makes it possible to transfer genes between/among different species. Stilbenes, a group of broad-spectrum phytoalexins, are poisonous to fungi, bacteria and virus. They play an important role at the early stage of plant defence system. Furthermore, stilbenes are beneficial to people. Stilbene synthase, the key enzyme in the biosynthesis of stilbenes in groundnuts, coverts one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA which are commonly exist in all plant species into 3,4',5-trihydroxystilbene in one step of reaction. Thus, stilbene synthase gene from groundnut was cloned and transferred to elite maize inbred lines utilizing gene-engineering techniques in the present research in the hope of enhancing resistance in maize and improving maize quality. 899bp promoter sequence(ubi-1), together with 83bp untranslated exon and 1010bp first intron sequence of maize polyubiquitin was cloned by the technique of PCR Amplification. When sequence of the cloned ubi-1 was compared with the corresponding region of the published polyubiquitin sequence, 3 out of 899bp were different, and these three mutation sites did not occur at the sites of functional elements such as TATA box. The rest of the cloned sequence was 98.69ï¼… in common with the published one in the corresponding region. Then, the promoter and 5'UTR were fused with GFP gene and the expression framework of ubi promoter-5'UTR- GFP-Nos terminator was constructed. At last, Particle bombardment procedures were hired to transfer Res gene to onion epidermis cells and 16-18 hours after bombardment, green fluorescence was seen in the cells with fluorescence microscope. This confirmed that the promoter was functional and the 5'UTR had the ability to enhance gene expression.Stilbene synthase gene of ground nuts was cloned by RT-PCR procedures. Sequencing analysis demonstrated that the cloned Res gene shared a 93.25% homology with the published STS gene in GenBank. When the deduced amino acids sequences of the cloned Res gene and those of the published STS gene were compared, 7 out of 389 amino acids were different. But none of the mutation occurred at the C164 active center of Stilbene Synthase. So the function of the synthase may not be affected.Plant expression vector of Res driven by ubi-1 and the 5' UTR of maize polyubiquitin gene was constructed based on pBI121 by replacing CaMV35S in pBI121 with the cloned promoter sequence and GUS with the cloned Res gene, respectively.Maize typeâ…¡calli from two different elilte inbred lines, Zong31 and Zheng22, were transferred by particle bombardment and the optimized parameters we gained are as follows: the amount of gold power per bombardment is 60ug; the optimal times of shotting is twice; and the most suitable shoot distance is 9cm. At the same time, regeneration system of maize were also studies in this work. Both embryo ages and hormone concentrations influence the calli induction ratio greatly. Embryos harvested 11 days after pollination were much easier to be induce into type â…¡ calli, while too old or too young ones would reduce the ratio of type â…¡ calli induction. Concentration of 2... |
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