Studies On Identification Of Low Copy Endogenous Genes Of Sugarcane And PCR Detection Technology

Low copy endogenous genes of sugarcane were identified referring to the existing endogenous reference genes of transgenic plants, known low copy genes and putative low copy genes according to their function. Twelve sugarcane cultivars were selected out to represent different genetic backgrounds of sugarcane cultivars in China and tested to verify copy number of the endogenous genes selected. PCR protocols were set up to detect the low copy endogenous genes subsequently, which lays the foundations for establishing detection standards of transgenic sugarcane.1. Based on the specificity and universality of endogenous reference genes,15 endogenous genes of sugarcane species were screened out after PCR analysis with DNA samples extracted from 12 sugarcane cultivars. The genes are ALS, P4H, ENOL, GR, PS, TST, APRT, CYC, PRR, F2KP, IPI, IVR, NCED,18S rRNA and 5S rRNA-ITS.2. Quantitative PCR were performed to the 15 endogenous genes with the 12 sugarcane DNA samples as templates and 4 genes, namely ALS、P4H、GR、PRR were proved to be low copy genes in sugarcane referring to known mean molecular weight of sugarcane.3. To verify the results of quantitative PCR, southern blotting experiments were carried out,with ALS gene as a sample. The results showed there was only one band on southern blotting membrane to every sugarcane genomic DNA, which further proved ALS gene has a single copy in sugarcane.4. A pair of primers were designed according to the sequence of ALS gene and a PCR protocol was set up to detect the gene. The 12 sugarcane DNA samples were tested and one specific band of 171 bp was produced in each of the 12 DNA samples.5. A quantitative PCR protocol was developed to detect ALS gene and a minimum 0.16 ng sugarcane genomic DNA produced the specific band of ALS gene. The quantitative PCR correlation coefficient of standard curve was 0.999 (R2) and the amplification efficiency of primer (E) was 0.950553.6. To test workability of the PCR protocol of ALS gene, PCR experiments were performed with genomic DNA extracted from samples of maize, rice, soybean and sugarcane. Only DNA samples from sugarcane produced the specific band of ALS gene, which indicates the PCR protocol of ALS gene is workable in sugarcane species identification.