Screening And Identification Of Transgenic Arabidopsis Thaliana Of MirXTH And EdMAN From Mirabilis Jalapa L.

The establishment of microbial-plant joint repair system and the mechanism of mutual restoration and repair of petroleum-contaminated soil have become the research hot spots in this field,while the pollution-resistance ability of plants has become one of the limiting factors for the establishment of the system.The cell wall is not only the first cell structure that senses external stress signals,but also the first layer of defense system against external stress to protect plant cells.Therefore,In our research group,the Mirabilis jalapa L.which have more develope roots and well tolerance in low and medium oil concentration is used as experimental materials,the two protein genes involved in cell wall rebuilded and reorganized,relaxation and ductility regulation: MirXTH and edMAN are used as experimental objects,study and reveals the molecular mechanism of the stress response of the roots of Mirabilis jalapa L.under oil pollution.The result of this study are as follows:1.The MirXTH and edMAN are transformed into wild-type Arabidopsis thaliana by agrobacterium-mediated flocculation infection.Using hygromycin screening and preliminary identification of PCR,we obtain twenty edMAN positive plants and five MirXTH positive plants.2.The extraction of protein from Mirabilis jalapa L.,wild-type Arabidopsis thaliana and homozygous MirXTH and edMAN positive plants by acetone method,while Mirabilis jalapa L.as positive control and wild-type Arabidopsis thaliana as negative control,after Western blot experiments,the exogenous genes,edMAN and MirXTH,successfully translated and finally identified thirteen and three transgenic lines contain edMAN and MirXTH.3.Using wild-type of Arabidopsis thaliana as a blank control,real-time quantitative PCR is used to analyze the expression level of MirXTH and edMAN in homozygous transgenic plants.The result showes that the MirXTH and edMAN are overexpress in Arabidopsis thaliana.4.We select lines with high relative expression and low relative expression of edMAN and MirXTH transgenic lines to observe the phenotype.Compared with wild-type Arabidopsis thaliana,the edMAN transgenic lines have the phenomena as follows: the number of the lateral roots,the length of the main root,the number of rosette leaf and bolting are significantly increase.the MirXTH transgenic plants have the phenomena as follows: the number of the lateral roots increase,the length of the main root increase too.According to the phenotypic observation of the selected transgenic plants,we found that MirXTH and edMAN improve elongation and growth of the root and leaf,and then reveals the function of MirXTH and edMAN in Mirabilis jalapa L..It also can lay a solid foundation for study the molecular mechanism of Mirabilis jalapa L.in repairing the root cell wall and paving the way for reveal the tolerance mechanism of Mirabilis jalapa L.at the molecular level under petroleum pollution condition.