Cloning And Expression Analysis Of Gene AhGLB Involving Waterlogging-tolerance In Peanut

China is one of the main peanut production、consumption and exporting countries in the world, and the annual average total output account for about40%of the world’s. Peanut will be suffered from the hypoxic stress caused by waterlogging due to the long-term rain or flooding, which seriously affects its yield and quality. In recent years, the morphology and yield construction of peanut tolerant to waterlogging、 the physiological and biochemical basis、 identification of germplasm resources and breed improvement have all made progress, but the molecular mechanism of peanut resistant to hypoxic stress caused by waterlogging is no clear. Studies have shown that when plants suffered to waterlogging stress it will produce high concentration of NO (free radical) and hypoxic stress which will causing multiple injuries to the cells. Recently non-symbiotic hemoglobin have been found in one after another in some plants, it can combine with NO to form NO3,which not only decreasing the concentration of NO in cell, but also provides the oxygen source, so as to reduce the free radical and hypoxic stress caused by waterlogging. In view of this, in this experiment we chose three peanut varieties which have different waterlogging tolerance as test materials, trying to clone the gene (Arachis hypogaea GLB, AhGLB) in peanut that encoded non-symbiotic hemoglobin, then determinate the expression of AhGLB in peanut roots、 stems and leaves using quantitative PCR, further lay the foundation for construction of justice expression vector conducting genetic transformation and detection the fuction of AhGLB gene in peanut resistant waterlogging. The conclusions are as follows:1、Found and downloaded the GLB coding sequences in many plants at the Genebank, compared and analyzed the five plants that have the highest similarity which are Arabidopsis thaliana、 Solanum lycopersicum、Solanum tuberosum、 Medicago sativa Linn and Glycine max to design degenerate primers, using it to clone the middle fragment of non-symbiotic hemoglobin gene from peanuts, and the length was351bp. Obtained AhGLB gene full-length613bp by RACE technology. Homology analysis of AhGLB gene showed that, gene with the Glycine max、 Vigna radiata、 Lotus japonicus and other legumes, the non-symbiotic hemoglobin gene homologous are highest, reach more than85%.2、 By designed primers for quantitative PCR on the AhGLB gene, conducted PCR reaction to get gene fragments of AhGLB and made of standard substance; using the diluted standard proceed Quantitative PCR again, build AhGLB gene quantitative PCR standard curve according to the different dilutions of the standard fluorescent quantitative. That provides an effective method for the determination of the amount of the peanut AhGLB gene expression. Using this method measurement the AhGLB gene expression in different peanut varieties、 organization and environmental conditions (oxygen、 nitrogen). The results showed that in normal oxygen condition the highest expression of peanut non-symbiotic hemoglobin gene AhGLB is in the root, followed by leaves and stems; hypoxic stress can induce the AhGLB gene expression in peanut, and the expression level in the roots is much larger than the blade parts, the expression level of genes in three varieties is:Xiang Hua2008>Yu Hua15>Zhong Hua8; induced by nitrogen(KNO3), the roots non-symbiotic hemoglobin gene AhGLB expression levels compared with controlled group, the content of changes first increase and then decrease; the leaves AhGLB of gene expression level increase at first and then reduce, the final rise again to show "N"-shaped trend, in varieties, compare with other two-species Xiang Hua2008have the differences expression, it is the largest, then followed by Yu Hua15and Zhong Hua8.Summarizing the above analysis,we can speculate preliminary,the gene by cloned associated to the resistance of peanut waterlogged anoxic stress.