Obtaining The Drought Resistance Transgenic Wheat By Using The Improved Minimal Expression Cassette Technology

With the biolistic transformation no host restrictions and non-seasonal time constraints, it’s easy to operate that accounting for more than65%of all genetically modified crops. Using the minimal expression cassette technology transgenic into plant, which only contains the promoter, coding sequence and terminator expression cassette sequence, to avoid the security risks that may be caused by the skeleton sequence without the excluding the vector backbone. The nuclear matrix attachment region sequences of SAR (the scaffold attachment region) is an important part of the eukaryotic nucleus, the mainly functions to dissemination of genetic information and transcriptional, which enhance the coordination of adjacent components, so that the gene fragment between the two SAR is defined as an independent ring structure of chromatin, thereby the SAR as the role to block the influence of neighboring chromatin areas can improve the target gene expression. In order to improve the stability expression of exogenous gene, this study add the SAR at both ends of the minimum expression cassette sequences to construct the expression vector, and establish a minimum expression cassette for wheat transformation. First, with GUS as a target gene, herbicide resistance gene Bar as marker genes, verified the pSGUS vector. On this basis, in order to create drought in transgenic wheat germplasm, get the target drought related transcription factor gene GmDREB3from soybean, Bar gene as the selecTable marker gene, biolistic transformation different receptors, such as Jimai22, Shi366, Shi B074056, Shi15, the results are as follows:1. Biolistic transformation GUS gene, gene gun bombardment Kenong199receptor,by PCR getting9positive wheats. Randomly selected four wheats (No. M2-1, the M2-2, the M2-3and M2-4) staining the GUS reporter gene, staining in the radicle. Further staining of the T1generation of the endosperm to the four wheats (randomly selected three wheats) and flowers (randomly selected three wheats) to detecte the GUS gene expressionion, found both of the endosperm and flowers can be detected GUS expression, indicating that use the improved minimal expression cassette into the sTable expression of exogenous gene in the recipient.2. Biolistic transformation GmDREB3gene to Jimai22receptor wheat, by PCR get30positive wheats, that randomly selected six positive wheats to RT-PCR, the results that exogenous gene GmDREB3can be expression at the transcriptional level, further by real-time PCR analysis of transgenic wheat’s copy number, the results show that the positive plants with low copy number. These results suggest that add the SAR both ends of the minimal expression cassette sequence can significantly improve the integrity and stability of the conversion fragments, to reduce the integration of the target gene copy number.3. To determine the different receptor the expression level of the target gene, and trans GmDREB3to different receptor such as Jimai22, Shi366, ShiB074056and Shi15receptor wheat, and the positive rates were as follows:0.53%,0.80%,0.99%and0.31%, indicating that the ShiB074056receptor get the highest conversion efficiency of different receptors, it’s indicating that the different receptor wheats also affect the expression level of minimal expression cassette with SAR.4. After the turn the gene GmDREB1materials receptor Jimai20MG3a, the MG7and MG14; Shi4185receptor transgenic GmDREB3materials of the MG2-3and the MG3-9; and JiMai19-receptor transgenic GmDREB3of drought physiological indexes in materials MG30-5POD and Pro content measurement, and the yield monitor, the results show that the MG3-9, whether under the dryland treatment or water treatment conditions, POD activity and Pro content is superior so receptors and other transgenic lines; Yield performance487.4Kg/666.7m2in the than in the control varieties yield13.08%to38.11%, ranking this first of all tested strains.