Study On Genetic Transformation In Arabidopsis Of Wax Synthetic Gene CsCUT1 And CsFATB From Glossy Mutant Of Navel Orange

The glossy Navel orange mutant (MT) has a remarkable characteristic of unusual glossy peel. It is useful to study this mutant to understand the molecular mechanism of citrus wax biosynthesis. Our previous studies showed that at different developmental stages, CsFATB and CsCUT1 gene expression is different between the wild type Navel orange (WT) and MT, and the MT CsCUTl cDNA squence is different from that in WT. In this study, WT and MT was used as the experimental material to study the role of two key genes of CsFATB and CsCUTl in wax synthesis by overexpression these genes in Arabidopsis using Agrobacterium-mediated method. The results show as follows:1.CsFATB and CsCUTl overexpression vector constructionIn order to validate the function of CsFATB and the of CsCUT1 gene, the overexpression vector of pCAMBIA1301-CsCUT1-W、PCAMBIA1301-CsCUT1-M and pCAMBIA1301-CsFATB was successfully constructed.2. Screening of Positive transgentic plants of Arabidopsis thalianaUsing Agrobacterium-mediated inflorescence immersion method, pCAMBIAl3 01-CsCUT1-W、pCAMBIA1301-CsCUT1-M and pCAMBIA1301-CsFATB were introd uced into Arabidopsis. The seeds of transgenic was sowed in MS culture medium c ontaining hygromycin to screen the positive plants. To further confirm whether the exogenous genes introduced into Arabidopsis, transgenic and wild-type Arabidopsis genomic DNA was extracted and used as the template for PCR detection. As a res ult,5 strains of PC AM 1301-CsCUT1-W transgenic plants,3 strains of PCAM 1301-CsCUT1-M transgenic plants,7 strains of PCAM1301-CsFATB-W transgenic plants were obtained. These results suggested that CsFATB and CsCUT1 played an importa nt role in plant wax biosynthesis because the wax ultrastructure and composition alt ered in transgenic plants overexpressed these genes.3 Phenotypic analysis of positive transgenic ArabidopsisTB staining showed that T2 lines of PCAM1301-CsCUT1-W, PCAM1301-CsCU T1-M and PCAM 1301-CsFATB transgenic plant leaves were stained lighter than wil d type. Chlorophyll permeability experiment suggested that the chlorophyll permeati on rate of transgenic plant leaves were much lower than wild type at the same tim e point. Electron microscopy revealed that leaf and stem surface of all transgenic plants had less wax crystals than wild type.