| Maize(Zea mays)is the largest crop in China,and can be used as food,feed and industrial raw material.Increasing maize yield and improving maize quality are of great significance for agricultural production and industrial development.Compared with the traditional breeding technology,transgenic technology has the great advantages in enhancing crop resistance,increasing crop yield,and improving crop quality.The establishment of an efficient maize transformation technology system is the prerequisite for maize transgenic breeding.Based on the previous work,we optimized Agrobacterium-mediated maize transformation technology.The main results are as follows:Maize immature embryos were infected with Agrobacterium after heat treatment for 2min at 45 ?,which significantly increased the infection efficiency of Agrobacterium tumefaciens.Adding 100 mg/L cysteine in the co-culture medium can also improve the infection efficiency.By using two methods,the infection efficiency of Agrobacterium in maize inbred line Z31 and hybrid Hill can reach more than 90%.ELISA analysis showed that the content of active oxygen species(ROS)in cysteine-treated immature embryos were significantly increased,indicating that cysteine could increase the infection efficiency of Agrobacterium not only as an antioxidant,but also by other ways.The transcriptome sequencing analysis showed that 2795 genes were up-regulated and 2150 genes were down-regulated in Hill immature embryos treated with cysteine;1277 genes were up-regulated and 949 genes were down-regulated in Z31 immature embryos treated with cysteine;939 genes were up-regulated and 549 genes were down-regulated in both Hill and Z31.Hill had more changes in gene expression than Z31,indicating that Hill is more sensitive to cysteine treatment.The GO analysis showed that most of the up-regulated genes were related to the metabolic process,redox process and the activity of oxidoreductase,and most of the down-regulated genes were related to the membrane metabolism and the membrane integrity.Mapman analysis found that many genes are involved in cell wall metabolism,and the up-regulated genes are mainly glucan transferase genes,and the down-regulated genes are mainly expansion genes and fibroin synthesis related genes.The change of these genes may affect the metabolic balance of cell wall,increase cell wall permeability,and facilitate the invasion of Agrobacterium,so as to improve the infection efficiency.Agrobacterium-mediated maize transformation system was systematically optimized.Optimize culture conditions,embryos grown on the culture medium containing 6-BA and Cu2+,can grew to green callus with high regeneration ability under the condition of weak light culture(10-30?mol ·m-2·s-1),improving the selection efficiency and shortening the culture cycle.Optimize medium composition,by adding Cu2+in the co-culture medium and adding 1.5mg/L 2,4-D in the selection medium,can significantly improve the transformation efficiency of maize immature embryos.It was found that different vector skeletons affect transformation efficiency.The ZmBBM1 gene was artificially synthesized and sub-cloned into the plant expression vector pCAMBIA3301 and transformed the maize inbred line B73,Z31 and CAL,respectively.It was found that ZmBBM1 can significantly improve the transformation efficiency of maize.It can be used for the genetic transformation of stubble type maize inbred lines to expand the genotype of maize transformation receptors.This study systematically optimized maize genetic transformation technology,and constructed a set of highly efficient maize transgenic technology system,which provides a guarantee for gene function verification and the creation of new genetically modified maize materials with breeding value.At the same time,the molecular mechanism of cysteine enhancing infection efficiency is investigated,which provides a theoretical reference for further research on plant genetic transformation. |
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