Establishment Of An Efficient Plant Regeneration System From Peanut And Genetic Transformation Of Gene AhGLB

Peanut (Arachis hypogaea L.) is an important economic and oil crop, and China is one of the main peanut production consumption and exporting countries in the world. If peanut is suffered from the hypoxic stress caused by waterlogging due to the long-term rain or flooding, this will seriously affect its yield and quality. In recent years, some research results found that non-symbiotic hemoglobin in plants can combine with NO to form NO3-, which reduces hypoxic stress caused by waterlogging. The aim of our research is to obtain a better insight into the molecular function of AhGLB in response to abiotic stress. At the base of cloned AhGLB gene from peanut, we have constructed of sense> anti-sense expression vector, and establishment of efficient regeneration system of peanut. Optimization of peanut genetic transformation technology system, through PCR and southern blot analysis we could figure out that AhGLB gene was successfully transferred to tobacco and peanuts. Measured by quantitative real-time PCR conducting genetic transformation and detection the fuction of AhGLB gene in peanut resistant waterlogging. The conclusions are as follows:1. By Yuhua15, Xianghua2008, Zhonghua8and Zhonghua4conducted in different parts and different hormones bud induction medium optimal screening test, we established peanut efficient regeneration system. Experimental results show that different genotypes have different regenerative capacity. Adventitious shoot formation of Xianghua2008and Zhonghua8the highest percentage of regeneration is up to86%. Based on these experiments, we choosed Xianghua2008and Zhonghua8for stem elongation and rooting experiment. Xianghua2008and Zhonghua8respectively the suitable concentration of6-BA is4mg/L and2mg/L; root formation in MS medium with NAA concentration of1.5mg/L and2mg/L is best.2. Successfully constructed AhGLB gene overexpression and inhibition of expression vectors and transferred to the Agrobacterium EHA105, lay the foundation for genetic transformation of genes. Research on AhGLB gene genetic transformation experiments shows:Different peanut varieties have differences on kanamycin tolerance; The feltration medium supplemented with200mg/L or20mg/L Km, can efficiently inhibit shoots regeneration or rooting from peanut.3. To obtain transgenic peanut. Through the methods of PCR and Southern blot analysis to preliminary determination that AhGLB gene was successfully transferred to plants. By quantitative real-time PCR experiments measured that sense transgenic plants expressing the amount of AhGLB gene are significantly higher than the wild-type plants.