Cloning And Functional Analysis Of ZmEREB87 Transcription Factor Gene In Maize(Zea Mays L.)

Maize(Zea mays L.) is one of the most widely distributed food crops in the world, an important economic crops, livestock feed and industrial raw materials, which its planting area had more than wheat, rice and became the first major food crops in China. However, many environmental factors, such as drought, low temperature, high salinity, water logging, disease and others, have a serious impact on the growth and development of maize, cause great losses in agricultural production at the same time, and it is the direct reasons of corn yield reduction. With the further study of molecular biology and functional genomics, mining the key resilience resources and cultivating new crop varieties with the use of genetic engineering or molecular markers has become a new direction of plant breeding work.According to the results of the drought transcriptome sequencing of strong droughtresistant maize inbred lines F83 and selected the AP2/EREBP transcription factor family members Zm EREB87 induced by drought and significantly increased expression as the research object, and cloned the gene from F83 used RT-PCR, analyzed the basic characteristics of that gene and evolutionary relationships by the method of bioinformatics, studied the tissue-specific and induced expression patterns in different abiotic stress of Zm EREB87 using real-time fluorescence quantitative PCR(real-time quantitative PCR, q RT-PCR), built the plant overexpression vector and made a genetic trangformation to arabidopsis and tobacco respectively using the agrobacterium mediated, made a preliminary analysis of the function in drought-overexpressing line. The main results were as following:1. According to the m RNA sequence of maize transcription factor genes Zm EREB87 designed specific primers and cloned the gene Zm EREB87 from F83 by RT-PCR, the bioinformatics analysis showed that the gene encodes a 315 amino acid residues, and the Zm EREB87 protein is an unstable protein with 63.03 unstable index, 33.8 KDa theoretical molecular weight, 5.25 isoelectric point; conserved domain analysis showed that Zm EREB87 belongs to EREBP transcription factor ERF subfamily members; phylogenetic analysis indicated that Zm EREB87 had higher relationship with Sb EREBP protein in sorghum and further relationship with At ERF07 protein in arabidopsis and Vv EREBP protein in grapes.2. Using q RT-PCR method to analyze the genes tissue-specific and induce expression patterns in six kinds of abiotic stress. Results of tissue-specific showed Zm EREB87 gene were expressed in young shoots, silk, primary roots, ear, ear leaf, bract leaf and tassel, but the highest expression in the young shoots; three-leaf stage of maize seedlings were treated by six kinds of abiotic stress, we found that Zm EREB87 gene was induced by ABA(100μM), dehydration drought, high salt(250 m M Na Cl), 20% PEG, high temperature(42℃) significantly and inhibited by low temperature(4℃), which indicated that Zm EREB87 gene may involved in the response to abiotic stresses process by ABA-dependent signal transduction pathways.3. The p CAMBIA3300 was used as the basic vector, we conducted a vector p CAMBIA3300-K containing a Kpn I site using the polymerase chain reaction(PCR) technology. Then we constructed the plant expression vector p CAMBIA3300-35S-EREB 87-NOS containing the gene Zm EREB87 by constructing intermediate vector p CAMBIA 3300-K-EREB87, and obtained engineering bacteria using agrobacterium tumefaciens LBA4404. Tobacco and Arabidopsis thaliana was disseminated genetic transformation through Agrobacterium inflorescence and leaf disk method, the preliminary evidences showed that the foreign genes had been introduced into the plant genome.4. We observed the changes of phenotypic in transgenic tobacco through drought treatment. The results show that transgenic line and wild strain all wilting after drought, and wild lines have worse than transgenic,which preliminarly indicated that transgenic palnts has stronger drought resistance than wild. And we analysized the feature of physiological characteristics under the treatment of 15% PEG were employed to stimulate drought, the results showed that the relative water content(RWC), proline(Pro), soluble sugar content and superoxide dismutase(SOD), peroxidase(POD), catalase enzyme(CAT) antioxidant enzymes activities of transgenic lines were higher than the wild strain, and relative conductivity and malondialdehyde(MDA), hydrogen peroxide(H2O2) content is less than the wild strain, furtherly indicated transgenic plants have more strong drought resistance.5. We observed the changes of phenotypic in transgenic arabidopsis through drought rehydration treatment. The results show that transgenic line and wild strain all wilting after drought,but only transgenic lines returned to normal after rehydration,which preliminarly indicated that transgenic palnts has stronger drought resistance than wild. And we analysized the feature of physiological characteristics under drought treatment, which indicated that the RWC, Pro, soluble sugar content and SOD, POD, CAT activities of transgenic lines were higher than the wild strain, and relative conductivity, MDA, H2O2 content is less than the wild strain, furtherly indicated transgenic plants have more strong drought resistance.