Cloning, Characterization And Genetic Transformation Of CitSERK Gene In Citrus

The competence for somatic embryogenesis of callus with long-term conservation isalways the hot problem in plant cell engineering. Some calli still have strong competencefor somatic embryogenesis after a long time. While that of some others decreased rapidlyor almost completely lost this capacity. Cloning the genes associated with the somaticembryogenesis of citrus callus is important for plant regeneration following callustransformation. Studies on somatic embryogenesis of citrus callus will help understandthe molecular mechanism of somatic embryogenesis and improve the efficiency of citruscallus utility.In this study, 'Anliu' sweet orange was used to investigate the mechanism of somaticembryogenesis. Full cDNA of CitSERK from Anliucheng callus was amplified byRT-PCR. Its nucleotide and putative amino acid sequence were analyzed. Meanwhile, thesense and anti-sense plant expression vectors were constructed. At present, the genetictransformation has being done. The main results are as follows:1. Total RNA from Anliu callus was extracted. The rate of A₂₆₀/A₂₃₀ of RNA was over2.0, and the A₂₆₀/A₂₈₀ranged from 1.65-1.90, indicating low levels of polysaccharideand good purity of the RNA. Its quality was fine for subsequent molecular biologyexperiment.2. The Full cDNA of CitSERK was amplified by RT-PCR, and it has 1886 nucleotide. Atthe same time, the recombinated plasmid DNA was tested by PCR and enzyme digest.3. CitSERK was analyzed by BLAST, EXPASY and Clustal W on internet, it has highhomology score at amino acid level with other SERKs. Results showed that thenumber of putative amino acid is 621, isoelectric point is 5.57 and molecular weight is68.3kD. It is a hydrophilic amino acid.4. Sense and antisense expression vectors of CitSERK were constructed. The Sense andantisense recombinated plasmid DNA was tested by PCR and enzyme digest.5. Transgenic Anliu callus were obtained by the method of Agrobacterium-mediatedtransformation.