GmMIOX Gene From Soybean Resistanted To Soybean Cyst Nematode Expression Analysis And The Construction Of Its Over Expression Vector

The Soybean Cyst Nematode disease is one of the serious disease that decreasing the production of soybean in the world today. Therefore, the study on the Soybean Cyst Nematode disease has been gaining increasing attention in the group of governments and researchers. Agricultural and chemical control measures have many problems, so nematode resistant base for genetic engineering become hotspot of research at present, cloned one or a plurality of resistance gene vectors transformed into plant, which enables the plant to produce a stronger, more durable resistance, in order to achieve a defense.Inositol oxygenase gene was found in the soybean genome, which is named for GmMIOX, by transcriptome sequencing, found that the gene before and after the Soybean Cyst Nematode infection appear significantly different expression, suggesting that the gene may be associated with soybean to Soybean Cyst Nematode resistance. However, there is no relevant studies has been reported in the field of soybean’s MIOX gene, much more studies of MIOX gene are focused on the research field of animals. Therefore, the aim in this study was to investigate whether the gene plays GmMIOX resistance in infection under SCN3, and to verify its function.There are two kinds of soybean in the research of resistant and sense of disease, Huipizhi Heidou and Liaodou 15, using RT-PCR screened GmMIOX gene family may exert resistant effect on a gene as the main object of study and by real-time quantitative PCR detecting its before and after expression changes in different periods of SCN3 inoculated and constructed over expression vector. The main results are as follows:1.The results showed that the nematode can invade the resistant varieties Huipizhi Heidou, but the number of nematode invasion huipizhi Heidou is less than Liaodou 15, in Huipizhi Heidou third instar larvae and adult numbers significantly less than Liaodou 15 that Huipizhi Heidou to nematodes is resistant to the development of anti-propagation effects.2.Detected by RT-PCR, it is found that GmMIOX4 gene in Liaodou 15 and Huipizhi Heidou are specific amplified product with obvious strip, and differences in the expression, it can be inferred that the possible effect of resistance to Soybean Cyst Nematode disease, so we choose the final selection of gene GmMIOX4.3.By real-time quantitative PCR method identified GmM10X4 gene expression changes in different periods, the amount of different varieties before and after inoculation of SCN3. In the absence of SCN3 infection, the relative expression of Liaodou 15 gene of GmMIOX4 in the root volume decreased gradually, while GmMIOX4 based Huipizhi Heidou root for relative expression without obvious the regulation, appeared in the 15 dpi when the relative expression amount reached highest point; under SCN3 infection, Liaodou 15 roots in the relative expression of GmMIOX4 gene is very low, until 25 dpi after inoculation, the relative expression of GmMIOX4 gene and GmMIOX4 gene increased, Huipizhi Heidou root inoculation of previous SCN3 relative expression low relative expression of GmMIOX4 gene in inoculated when 10 dpi reached the highest level, that is likely to be in the 10 dpi, the GmM10X4 gene plays resistance role, make the syncytial structure changed, nematodes can not rely on the syncytium absorb enough growth, the development of the nutrition, so that Huipizhi Heidou to suppress the role of nematode development, reduce the Huipizhi Heidou root number of adult, effect of resistance, or through the root epidermis of stage migration in the Soybean Cyst Nematode, which is blocked in the cells within the vascular bundles in the process of moving, play a disease resistant effect.4. From the Huipizhi Heidou by RT-PCR in CDS zone obtained GmMIOX4 gene, cloned into pGEM-T easy vector. The resulting sequence GmMIOX4 as materials, in the sequence ends with double enzyme cutting sites. After double enzyme digestion and plant binary expression vector pCAMBIA1303 connection, construction pCAM-MIOX4 over expression vector and the GmMOX4 gene, stable expression, which can be transformed into the plant cells, in the determination of SCN3 infection rate, therefore, to build a successful carrier for further study of its function and lay a solid foundation.