| Drought is one of the main limiting factors which are longly influence the world food production. It is predicted that the world population in2050will be over9billion, so, population and resources can be a quite prominent contradiction for a long time in future. With the development of modern genetics and molecular biology, breeding drought-tolerant crops using genetic engineering is imperative. Identification and cloning of new genes, can help understand the molecular mechanism of drought tolerance, create drought-resistant germplasm resources of genes, and breed drought resistance crops.Maize is not only an important food and feed crops, but also the important food industrial raw material and energy plant. In China, maize is the second largest food crops after rice. In this study, some stress responsive genes from maize excellent inbred lines X178were selected and drived from drought tolerant related ESTs, which had been obtained by cDNA-AFLP in our laboratory previously. Two new genes, a heat shock protein gene ZmHSP90-1and a transcription factor ZmMYB-R1were cloned. Through the bioinformatics analysis, protein instantaneous expression and quantitative real-time PCR, we studied initially response mechanism to abiotic stress of the two genes; We also analysised gene function by over-expressing in Arabidopsis. Our results are mainly as follows:1)72drought resistance related ESTs were analysised homology comparison and function in GenBank database,60ESTs have the homologous gene, most of these fragments were found to be associated with responsive genes to abiotic or biotic stresses. Whatis more, we discovered two new genes:a heat shock protein gene ZmHSP90-1and a transcription factor ZmMYB-R1. Other ESTs can’t find matching gene, the molecular techniques such as RACE will be used to clone the full length and analysis their biological function.2) The heat shock protein gene ZmHSP90-1, code697amino acids containing ATPase sites and HSP90conservative structure domain. Protein comparison and cluster analysis show that, there are a lot of highly conserved heat shock protein gene with ZmHSP90-1in Arabidopsis, rice and other species. The comparability of ZmHSP90-I and AtHSP90.1reached88.3%. Protein instantaneous expression analysis showed that ZmHSP90-1express in the cytoplasm. Quantitative real-time PCR analysis revealed that, the expression of ZmHSP90-1is affected by many kinds of abiotic stress. But under hot and salt stress, the expression pattern of the gene is similar, which deduced both shared common mechanism between the two stress responses.3) The recombinant vector of overexpressed ZmHSP90-1in plant was constructed and transformed into Arabidopsis by Agrobacterium tumefaciens-mediated inflorescence infection method. We obtained14T3generation transgenic lines by PCR and selected repeatedly by hygromycin. Seed germination and root length of overexpressed ZmHSP90-1Arabidopsis were restrained by NaCl, seedling and clonogenic assay displayed that the transgenic lines obtained inordinately stress tolerant to hot, drought, ABA and CaCl2, but under NaCl stress the performance of different lines is not consistent.4) The transcription factor ZmMYB-R1gene code626amino acids, which is R1-MYB type transcription factor containing a single conserved MYB-like DNA-binding domain. Protein comparison and cluster analysis show that the relationship of ZmMYB-R1and AtMYB3,OsMYB2is near. Protein instantaneous expression shows ZmMYB-R1expressed specifically in cell nuclei. Quantitative real-time PCR analysis revealed that the expression of ZmMYB-R1were induced by hot, salt, ABA, cold, drought. The recombinant vector of overexpressed ZmMYB-R1in plant was constructed and transformed into Arabidopsis by Agrobacterium tumefaciens-mediated inflorescence infection method. We obtained42T1generation transgenic lines by PCR and selected repeatedly by hygromycin, which will be further screened and carry function identification. |
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